Biostat B: Operating Manual [PDF]

Zitiervorschau

Operating Manual

BIOSTAT® B

BAE_B Rev. 2.32 - 0400 SW 4.1

Operating Manual BIOSTAT B

Introduction

Introduction This Manual applies to B. Braun Biotech International's bench-top fermentor system BIOSTAT® B, which is one example of B. Braun Biotech International's product program of sophisticated fermentation and peripheral laboratory equipment. For further information about this fermentor system and our complete product program please contact


B. Braun Biotech International GmbH Schwarzenberger Weg 73-79 D - 34212 Melsungen, Fed. Republic of Germany phone +49 56 61 - 71 34 00, fax +49 56 61 - 71 37 02 e-mail: [email protected] WebSite http://www.bbraunbiotech.com

or your local sales representative of B. Braun Biotech International GmbH.

Guide Through this Manual The Operating Manual is structured as shown below. It is not necessary to read this manual from the beginning. Especially experienced users can go straight on to the section of their interest. Section

:

Contents

1

:

Overview on design and equipment of the fermentor

2

:

Delivery and warranty information, installation and connection

3

:

Start-up and operation of the fermentor

4

:

Maintenance, assembly and equipment of the culture vessels

5

:

Detailed operating information about the digital control unit

6

:

Technical data

7

:

Supplement with additional documentation like technical drawings, etc.

Documentation Release Notes The Operating Manual refers to the design and equipment of the fermentor valid for the rev. no. and at the date of print. However, the characteristics and specifications of the BIOSTAT® B are subject to change. B. Braun Biotech International GmbH reserves the right to modify the equipment and to change the operating information without notice. B. Braun Biotech International GmbH assumes no obligation regarding design, ordering data resp. future manufacture unless otherwise agreed to in writing. The information given herein has been carefully estimated. However, this document is not necessarily complete. If you happen to find errors or if you miss specific information concerning special parts and their setup, please do not hesitate to contact your B. Braun Biotech representative or B. Braun Biotech International GmbH directly. B. Braun Biotech International GmbH

BAE_B Rev. 2.31 - 1099 SW 4.1

F190101 Return of Material

Declaration about decontamination and cleaning of equipment and components When returning equipment or components, please describe on page 2 of this form the problem(s) or fault(s) you have found. Please also indicate the remedial actions you require. To protect our personnel, we require all equipment or components be free of biological, chemical, or radioisotopic contaminants. We will only accept such equipment or components when: • •

the equipment or components have been adequately CLEANED and DECONTAMINATED. this declaring document has been completed, signed and returned by an authorized person.

Please help us in assuring a safe, hazard-free work environment.

A. Description of the Equipment or Component(s) Description / Cat. No.: ........................................................................................................................ ........................................................................................................................................................... Serial no.: ...................No. of invoice/delivery note .................................Date of delivery:.................

B. Contamination / Cleaning Attention: Please specify exactly the biological, chemical, or radioisotopic contaminant. The equipment was contaminated with:........................................................................................... ......................................................................................................................................................... ......................................................................................................................................................... Attention: Please describe the cleaning and decontamination procedure/method. and it has been cleaned and decontaminated by:............................................................................ ......................................................................................................................................................... .........................................................................................................................................................

C. Legally binding declaration I (we) certify that all information given in this form is correct and complete. The equipment and components have been adequately decontaminated and cleaned according to the legal requirements. No chemical or biological or radioisotopic risks remain that can endanger exposed persons' safety or health. Company / Institute: ......................................................................................................................... Address / Country:............................................................................................................................ ......................................................................................................................................................... Tel. / Fax (with area code):........................................................./ .................................................... Name of the authorized person: ....................................................................................................... Position:............................................................................................................................................ Signature / Date: ................................................................................................. /...........................


F190101E.DOC / Rev. 3 / 11.05.2000

Page 1 of 2


BBI-Dr. Wieneke

F190101 Return of Material D. Reason for return
wrong delivery


exchange


repair


modification


other

E. Please describe the problem(s) or fault(s) you have found (for repair) and/or indicate the remedial actions you require. ............................................................................................................................. ............................................................................................................................. ............................................................................................................................. ............................................................................................................................. ............................................................................................................................. ............................................................................................................................. ............................................................................................................................. ............................................................................................................................. ............................................................................................................................. ............................................................................................................................. .............................................................................................................................

F. Vom BBI Service auszufüllen (reserved for BBI service) Bemerkungen: ..................................................................................................... ............................................................................................................................. ............................................................................................................................. ............................................................................................................................. ............................................................................................................................. Please pack the equipment properly and send it to your local service representative or to B.Braun Biotech International, Germany (carriage paid to receiver).


F190101E.DOC / Rev. 3 / 11.05.2000

Page 2 of 2

B.Braun Biotech International Technischer Service Schwarzenberger Weg 73-79 34212 Melsungen Germany


BBI-Dr. Wieneke

Operating Manual BIOSTAT B Contents

Contents

Page

Introduction Guide Through this Manual Documentation Release Notes Declaration about Decontamination and Cleaning of Equipment and Components

1

Design and Function

1.1 1.1.1 1.1.2 1.1.3 1.2

Control Unit Thermostate System Drive System Air and Gas Supply Culture Vessels

1-1 1-2 1-2 1-2 1-3

1.3

Integrated Digital Control System

1-4

2

Delivery and Installation

2.1

Unpacking and Checking Completeness of Delivery

2-1

2.2

Warranty Agreements and Customer Service

2-1

2.3

Customer Service

2-2

2.4 2.4.1 2.4.2 2.5 2.5.1 2.5.2 2.5.3 2.5.4

Installing the Fermentor System Space Requirements P&I - Diagram Connection Specifications Connectors, tubings, cables and Fittings Mains Connection Laboratory Connection for Cooling Water Supply and Return Connection of Air or Gas Supply

2-3 2-3 2-3 2-5 2-5 2-5 2-6 2-7

3

Starting and Operating the BIOSTAT® B

3.1 3.1.1 3.1.2 3.1.3 3.1.4

Preparing and Connection of the Control Unit Preparing the Workplace Connecting the Thermostate System Connecting the Air/Gas Supply System Preparing and Connection of Peripheral Equipment for Measurement and Control Preparing the Culture Vessels and Accessories Checking the Equipment Assembling and Equipping the Culture Vessels Filling the Jacket of the Culture Vessel Filling the Culture Vessel Preparing and Connecting Addition Agent Bottles Sterilization of the Culture Vessel

3.2 3.2.1 3.2.2 3.2.3 3.2.4 3.2.5 3.2.6

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3-1 3-1 3-1 3-1 3-1 3-2 3-2 3-2 3-3 3-3 3-4 3-4

1

Operating Manual BIOSTAT B Contents

3.3 3.3.1 3.3.2

3.4 3.4.1 3.4.2 3.4.3 3.4.4 3.5 3.5.1 3.5.2

Peparing a Fermentation Run Overview Final Setting-up and Connection of the Culture Vessel for Fermentation Connecting the Probes and Setting the Measurement and Control System Carrying-Out a Fermentation Run Sterility Test Inoculating the Culture Vessel Adding Sterile Solutions during the Fermentation Run Taking Samples End of a Fermentation Run Harvesting the Product Cleaning and Maintenance

4

Equipment of the Culture Vessels

4.1 4.1.1 4.1.2 4.2 4.2.1 4.2.2 4.2.3 4.3 4.3.1 4.3.2 4.3.3 4.3.4 4.3.5 4.3.6 4.3.7 4.3.8 4.3.9 4.3.10 4.3.11 4.3.12 4.3.13 4.3.14 4.3.15 4.3.16 4.4 4.4.1 4.4.2 4.4.3 4.4.4 4.4.5

General Information Delivery and Starting up Inspecting a Culture Vessel and its Accessories Culture Vessels Dimensions and Design Features of the Culture Vessels Set-up, Equipment and Accessories of the Culture Vessels Top plate Design and Placement of the Accessories Equipping and Assembling the Culture Vessels Overview Disassembling a Culture Vessel Assembling a Culture Vessel Fitting of the Standard Equipment inside a Culture Vessel Baffle Insert and Baffles for Culture Vessels with Stirring Device Sparger Pipe Inoculation Connectors (Septum Port Kits) 1-Channel Inoculation Kit Universal-Adapter Quadruple Addition Port B Reference Thermometer Harvesting Pipe Harvest Filter for Removal of Medium at Microcarrier-Cultures Spinfilter Silicone Membrane Insert Blind Closures Probes and Electrodes General Notes Pt-100 - Sensor for Temperature Measurement Antifoam and Level Probes pH - Electrode pO2 - Electrode

3.3.3

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3-5 3-5 3-5 3-7 3-8 3-8 3-8 3-9 3-9 3 - 10 3 - 10 3 - 10

4-1 4-1 4-1 4-2 4-2 4-6 4-9 4 - 10 4 - 10 4 - 11 4 - 11 4 - 12 4 - 13 4 - 14 4 - 15 4 - 16 4 - 18 4 - 18 4 - 19 4 - 20 4 - 21 4 - 22 4 - 24 4 - 26 4 - 27 4 - 27 4 - 27 4 - 28 4 - 30 4 - 32

2

Operating Manual BIOSTAT B Contents

4.5 4.5.1 4.5.2 4.5.3 4.5.4 4.5.5

External Equipment for Connection to the Culture Vessels Exhaust Cooler Sterile Filters for Air/Gas Supply and Exhaust Corrective Solution Bottles By-pass Sampler Kit Manual Sampling Kit

5

Control Unit

5.1 5.1.1 5.1.2 5.1.3 5.1.4 5.2 5.2.1 5.2.2 5.2.3 5.3

General Information Operating Behaviour Switching the BIOSTAT® B „Off“ and „On“ Power Failure Menu Structure Operation of the Control Unit General Operating Information Basic Menu Structure Operating Information Main Function „PROCESS VALUES“

5.4 5.4.1 5.4.2 5.4.3 5.5 5.5.1 5.5.2 5.5.3 5.5.4 5.5.5 5.5.6 5.5.7 5.5.8 5.5.9 5.5.10 5.5.11 5.5.12 5.6 5.6.1 5.6.2 5.6.3 5.6.4 5.6.5 5.6.6 5.7

Main Function „CALIBRATION“ Calibration of the pH - Electrode Calibration of the pO2- Electrode Pump Calibration Main Function „Control Loops“ Extent of Controller Functions of the BIOSTAT® B Controller Operation in General Parametrization of Controllers in General PID - Controller Optimization Temperature Control Stirrer Speed Control pH - Control pO2 - Control Foam Control Level Controller Controller for Substrate Feed SUBS1 und SUBS2 Airflow Controller Main function "Maintenance" Recorders Printers Utility Functions Measurement Ranges Manual Operation of Digital Outputs, Standard Manual Operation of Analog Outputs, Standard Alarm Messages of System Functions

5-7 5-7 5 - 11 5 - 14 5 - 16 5 - 16 5 - 17 5 - 17 5 - 18 5 - 19 5 - 21 5 - 22 5 - 24 5 - 27 5 - 29 5 - 30 5 - 32 5 - 33 5 - 33 5 - 34 5 - 37 5 - 38 5 - 39 5 - 40 5 - 41

5.8 5.8.1 5.8.2 5.8.3

Factory Settings General Information Factory Settings for Measurement Ranges of Process Values Factory Settings for Controller Parameters

5 - 42 5 - 42 5 - 42 5 - 42

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4 - 34 4 - 34 4 - 36 4 - 38 4 - 39 4 - 41

5-1 5-1 5-1 5-1 5-2 5-3 5-3 5-4 5-4 5-5

3

Operating Manual BIOSTAT B Contents

5.8.4 5.8.5 5.8.6 5.8.7

Example: pO2-Controller with Airflow - Massflow Controller and O2 - Enrichment Schematic Drawing „Pump Logic of DFC - Software“ Arrangement Plan Control Unit of BIOSTAT® B Assignment Plan for Interfaces of the Control Unit BIOSTAT® B

6

Technical Data

5 - 43 5 - 45 5 - 46 5 - 46

6.1

Dimensions

6-1

6.2

Installations

6-1

6.3

Control unit

6-1

6.4 6.4.1 6.4.2 6.4.3 6.4.4 6.4.5 6.4.6 6.5

Culture Vessels 6-2 Culture Vessel Type B 2 6-2 Culture Vessel Type B 5 6-4 Culture Vessel Type B 10 6-6 Culture Vessel B 10, Special Version for Small Working Volumes 6 - 8 Culture Vessel B 2 - Airlift 6-9 Culture Vessel B 5 - Airlift 6 - 11 Additional Equipment 6 - 13

6.6

Consumables

6 - 13

6.7

Control Unit

6 - 14

7

Optional Equipment and Accessories This section intentionally left free in this version of the manual.

8

Supplement

8.1

Conversion Table for Degrees of Hardness of Water

8.2

Characteristic Values for Gases

8-1

8.3 8.3.1 8.3.2 8.3.3 8.4 8.4.1 8.4.2

Calculation Methods for Flowrates of the Rotameters Sizing of Flowmeters / Rotameters Calculation Method Manufacturer's Examples for Sizing Calculations Additional Drawings and Flow Charts P & I - Diagram of the BIOSTAT® B Drawings and Flow-Charts of Fermentor Options or Customized Fermentors

8-2 8-2 8-2 8-3 8-6 8-6

BAE_B Rev. 2.31 - 1099 SW 4.1

8-1

8-6

4

1

Operating Manual BIOSTAT B

Design and Function

1

DESIGN AND FUNCTION

The BIOSTAT® B is a compact, high quality laboratory fermentor system for small scale fermentation applications. It is especially adapted to the demands of biotechnology research in universities and in industrial research laboratories. The BIOSTAT® B consists of the following main components :


Compact control unit with integrated digital measurement and control unit.




The digital measurement and control unit for comfortable control of the BIOSTAT® B. Its functions are specially adapted to the requirements of economic fermentor operation.




Stirrer driven and airlift type culture vessels offering these features: – Jacketed glass vessels – Working volumes of 2 l and 5 l (all vessel types), optional of 10 l (stirrer driven type only) – Internal concave bottom section for optimum mixing even at low stirring speeds




For comfortable handling and transport the vessels are installed in a support which also carries holders for the storage bottles of addition solutions. A culture vessel will be placed besides the control unit, thus minimizing the space requirements of the entire unit.

1.1

Control Unit

The control unit contains the thermostate system and all installations required for power supply, the supply of cooling water, pressurized air and the waste water removal. All electrical components are placed in a separate part of the control unit and are protected against spray or splashing water.


The upper part of the control unit contains the operating terminal of the control system. It is designed in inclined arrangement for easy operation of the terminal.




The lower front panel of the control unit includes the mains control switch, the fillthermostate switch and a flowmeter for the control of the air supply.




On left hand of the front panel four peristaltic pumps are built-in, providing short distance to either the storage vessels and the culture vessel connections for the tubig connections.




On the left side of the control unit, directed towards the side where the culture vessel is usually placed, all connectors for the supplies of the culture vessels are integrated : – Thermostate in-/outlet – Cooling water in-/outlet – Air inlet with additional O2 – inlet for O2-enrichment operation, air outlet – Exhaust cooler in-/outlet – Mains supply and fuse – Power supply to stirring motor – Sockets for electrodes (temperature, foam, level, pH and pO2) – Sockets for peripheral units, such as recorders, printers, Gasmix-unit or host computer.

BAE_B Rev. 2.31 - 1099 SW 4.1

1-1

Operating Manual BIOSTAT B

1 Design and Function

1.1.1

Thermostate System

The thermostate system is an open, pressure-free system. It includes a electric heater of 600 or 800 W heating capacity, which supplies the necessary energy to the system, and a solenoid valve for cooling water supply. The circulation pump 2.4 (see P&I diagram in section 2 and in the supplement) delivers water of the preadjusted temperature to the culture vessel. The operation temperature is set at the control panel. The digital measurement and control system ensures a precise and constant temperature control. The minimum temperature in the culture vessel is about 8 degC above cooling water temperature (for a culture temperature of up to 60 degC). Cooling water will only be supplied when the temperature needs to be decreased. This design of the BIOSTAT® B minimizes the cooling water consumption. Under usual operating conditions, i.e. a fermentation temperature below 50 degC, only a minimum amount of water will be spent. If tap water cannot provide sufficient cooling, external cooling water supplies can be connected. 1.1.2

Drive System

The drive sytem includes a 180 W DC electronic motor. This motor is directly mounted onto the stirrer shaft in the vessel top-plate via an elastic coupling. Usually two 6-bladed disk impellers are mounted to the stirrer shaft. However, the standard impellers may be replaced by optional stirrers of other design, if required, see section 4 for availability and mounting of different types of stirrers. The default speed range is 50 to 1.200 rpm. For special applications, such as tissue cell cultures, which are sensitive to shear forces and require gentle stirring, and if the tubing insert for bubble free gas supply is used, an optional slow speed drive is available (cat.-no. 884 470/4, speed range 20 ... 600 rpm, installation exworks only). The DC motor and speed control system are designed to guarantee a high speed constancy even in case of large torque variations, as may result from intensive gassing or by changes of the medium viscosity during the growth of the culture. 1.1.3

Air and Gas Supply

The BIOSTAT® B can be connected to any precontrolled laboratory gas supply installation. Required flow of air (or inlet gas) is up to 10 l/min at a max. pressure of 0.8 barg (11.6 psig). An additional O2 inlet connector allows for connection of a second gas containing oxygen. If no extra oxygen supply should be connected, the O2-inlet is combined with the air inlet by a corresponding pipe adapter. Optionally a Gas Mixing System is available for supply of a gas mixture of up to 4 gases (air, oxygenenriched gas, nitrogen or CO2, for instance). Please call B. Braun Biotech International GmbH for detailed information, if you require such a device. If your BIOSTAT® B is delivered with a Gas Mixing System, please note the corresponding Operating Manual. The flowrate required for aeration of the culture will be adjusted at a rotameter on the the front panel of the control unit. Membrane filters provide for a sterile gas supply and exhaust of the culture vessel. The air (or inlet gas) will be introduced into the culture broth via a sparger ring.

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1-2

1

Operating Manual BIOSTAT B

Design and Function

1.2

Culture Vessels

There are different types of culture vessels available for the BIOSTAT® B:


Stirrer driven culture vessels type B 2, B 5 and B 10 offering max. working volumes of 2 l, 5 l and 10 l, respectively




Airlift type culture vessels B 2 - Airlift and B 5 - Airlift with internal loop system offering max. working volumes of 2 l and 5 l, respectively.

The culture vessels are made of borosilicate glass and the standard stirrer driven vessels have a height / diameter ratio of about 2 : 1 (2.5 : 1 with the vessel B 10). All vessels are heated via the jacket. Connecting of the thermostate system to the culture vessel is made with special quick couplings. The inlet connector of the jacket (lower connector) has a self-closing male adapter. The upper outlet connector has a female adapter which is open. This design ensures that the jacket is kept pressureless when the culture vessel is disconnected from the control unit and allows for pressure compensation with the ambient pressure when the vessel is autoclaved. Thus overpressure cannot occur in the jacket during autoclaving, which may destroy the glass vessel. The culture vessels type B 2 and B 5 have 4 integrated side entry ports in the head space for access to the interior culture vessel. Through these side entry ports, corrective solutions can be supplied to the culture during the fermentation run, for instance. The top-plates are mounted to the flanged ring of the glass vessels with knurled screws. Each topplate contains ports of 19, 12 and 6 mm for assembly of electrodes and accessories, see section 4 of this manual for further details. In addition 4 tubing connectors are welded into the top plate. The vessels have an internal concave bottom section. This is a vessel design which is proven in fermentations of microorganisms and in the cultivation of animal cells. All parts in contact with the medium are made of stainless steel (1.4571) and the O-rings consist of EPDM (ethylene propylene rubber). The culture vessels are mounted into a supporting frame. Two handles allow easy transport of the vessels. The holders of the storage bottles for addition solutions are welded to the bottom support. This allows easy assembling and comfortable handling. When the culture vessel is placed in the autoclave the max. height of the culture vessel together with all equipment and the exhaust cooler determines the required dimensions for the autoclave. On request the exhaust cooler is available with a flexible connection piece, which can be bent over. Fig. 1-1:

Culture vessel type B 2 of the BIOSTAT® B

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1-3

Operating Manual BIOSTAT B

1 Design and Function

1.3

Integrated Digital Control System

The BIOSTAT® B has an digital measurement and control system which offers a defined set of functions for a broad range of standard fermentation applications. The measurement and control system is integrated in the control unit. Operating data are entered at the functional keyboard of the operator terminal in the upper front panel of the control unit. Process data will be indicated on an easy to read alphanumerical display. The hardware consists of a highly integrated microprocessor board which carries the measurement amplifiers and actuator controls. The microprocessor system includes a 80C188 processor, 128 kByte RAM and 128 kByte EPROM. The board has 8 analog inputs, 8 analog outputs, 8 digital inputs and 16 digital outputs. The software offers all functions necessary for fermentor operation, such as measurement and control of the parameters, calibration of electrodes and the intergrated pumps and for the signal transfer from/to peripheral equipment, such as recorders, printers and host computer for further data processing. The software version which this manual refers to, is revision 4.1.1-1) Standard measurement and control functions are for temperature, foam, level, substrate supply, pHvalue, partial pressure of oxygen (pO2) and control of a gasmix unit. The extent of measurement and control functions implemented in a customized fermentor version can be different, according to the specifications aggreed to in the order. After power down the system automatically restarts with the settings before. If the interupt takes longer than an adjustable interupt time (FAILTIME), the system proceeds as with switching off by the mains switch, i.e. the system restarts with a definable state of operation.

1-1)

If you have a control unit with an elder or more actual software version, please contact B. Braun Biotech International GmbH for a corresponding version or update of the Operating Manual.

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1-4

2

Operating Manual BIOSTAT B

Delivery and Installation

2

DELIVERY AND INSTALLATION

2.1

Unpacking and Checking Completeness of Delivery

The BIOSTAT® B is delivered after a thorough functional test of all mechanical and electrical components. The test includes a sterility test of 100 h for which all the ordered mounting parts are installed. Packing is done only by qualified personnel. After receipt of the the BIOSTAT® B and in case is inoperative due to transport damage and in order not to forfeit possible warranty claims for such damage or other defects, please pay attention to the following advices : 1.

Check whether the delivery is complete according to your order.




The delivery includes all connectors, adapters, cables and tubings required for assembly and connection of the BIOSTAT® B. You must only these parts.




Never use parts and accessories, which do not fulfill the specifications of B. Braun Biotech International. In such a case our warranty will lapse. B. Braun Biotech International GmbH will not be liable for malfunctions and damages resulting from the use of parts which were not released for use with the BIOSTAT® B.

2.

Check carefully all parts for damages, especially the culture vessels. Never use a damaged glass culture vessel (even if only hairline cracks occur or damages are suspected).

Warning: A damaged culture vessel might burst during sterilization in the autoclave. 3.

2.2

If parts are missing or damages occurred in transit, please inform your local B. BRAUN sales representative or contact B. Braun Biotech International GmbH, Melsungen, directly.

Warranty Agreements and Customer Service

B. Braun Biotech International GmbH warrants its products according to the „General Terms and Conditions of Business“ and unless other terms were agreed upon in writing. Date of reference is the date of delivery, which must be evidenced by a corresponding delivery confirmation.


The BIOSTAT® B is designed for use under normal laboratory environmental conditions.

Note: Before a use under specific environmental conditions in the laboratory and/or the use together with special nutrients or additional solutions, you will have to test the applicability of all parts.


The warranty is granted against defects of construction, manufacture or in material and any malfunctioning resulting therefrom.




The warranty is neither granted for parts subject to normal wear and tear (O-rings, seals, membrane filters), nor for defects or damages caused by improper handling, nor for defects and malfunctions caused by corrosion (i.e. due to lack of resistance against solvents, etc., which are used for the fermentation).

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2-1

Operating Manual BIOSTAT B

2 Delivery and Installation

2.3

Customer Service

Defects and damages can be repaired by your local B. Braun Biotech International GmbH service center. Defective devices can also be returned to your local sales representative or to B. Braun Biotech International GmbH. Carriage charges will be payable by the customer. The repairs will be carried out and charged in accordance with our terms of maintenance.


For return of any equipment please note your local sales representative of B. Braun Biotech International GmbH or contact directly B. Braun Biotech International GmbH Schwarzenberger Weg 73-79 D - 34212 Melsungen, Fed. Republic of Germany phone +49 56 61 - 71 34 00, fax +49 56 61 - 71 37 02 e-mail: [email protected] WebSite http://www.bbraunbiotech.com

Note: On return the parts must be clean, in good hygienic condition and carefully packed. Contagious parts must be desinfected or sterilized, according to applicable chemical, biological, biotechnogical or genetic safety regulations. The sender has to prove compliance with releted safety regulations. The sender will be charged for repair of damages due to transport and for necessary cleaning and desinfection of any parts.


If repairs are to be carried out on the premises or in a customer-owned workshop by own technical personnel, the necessary spare parts can be bought from B. Braun Biotech International GmbH. A detailed list of spare and wearing parts is available on request.

Note: In no event will B. Braun Biotech International GmbH be liable for repairs carried out by the customer and possible damages resulting therefrom.

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2-2

2

Operating Manual BIOSTAT B

Delivery and Installation

2.4

Installing the Fermentor System

2.4.1

Space Requirements

The BIOSTAT® B is designed for bench-top use. In general the space requirements depend on the type and number of the components and peripheral equipment which should be connected. Due to the variety of possible configurations the space requirements can vary considerably. 1.

Check the working place, whether it offers sufficient space. It is recommended to consider some additional space for convenient handling of the equipment.

2.

A standard configuration of a BIOSTAT® B with culture vessel B2 requires a bench space of about W x D = 800 x 600 mm. You can vary the arrangement of the components according to the lengths of the connecting tubings and cables.




For the dimensions of the control unit see the fig. “Dimensions and connectors of the BIOSTAT® B. The dimensions of the culture vessels are shown in part 4 of this manual. The dimensions of additional equipment and peripheral devices are shown in the corresponding documentation of these devices.

3.

Verify that the working place can carry the weight of the fully equipped fermentor system with the culture vessel and all peripheral devices, ready for operation.

4.

Check whether all supplies and installations are available at the operating site, such as for power supply, air or gas supply, water supply and drain, or prepare them correspondingly.




Tubings, clamps and accessories required for connection are delivered with the fermentor system. For dimensions of the tubings and connectors see tables below.

2.4.2

P&I - Diagram

Fig. 2-1

P & I - Diagram of the BIOSTAT® B (example for standard design; a copy of the original drawing or of a P&I diagram for a fermentor system of customized design will be added to the supplement of this Operating Manual)

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2-3

2

Operating Manual BIOSTAT B

Delivery and Installation

Fig. 2-2: Dimensions and connections of the BIOSTAT® B Pos.

Connector

Use

Air

hose connector

air inlet from laboratory connection

Air

hose connector

air supply to culture vessel

O2 õ

hose connector or adapter combined with Air õ

extra inlet for oxygen source (O2-enrichment)

Exhaust cooler õ

hose connector

cooling water backflow from exhaust cooler

Exhaust cooler

hose connector

cooling water supply to exhaust cooler

Thermostate õ

tubing with female connector

cooling water backflow from double jacket

Thermostate

tubing with male connector

thermostate supply to culture vessel

Cooling water

hose connector

cooling water return to lab or drain

Cooling water õ

hose connector

cooling water supply from laboratory

pH

pH-electrode

connection of pH-electrode

Foam

Foam-probe

connection of antifoam probe

Level

Level-probe

connection of level probe

pO2

pO2-electrode

connection of pO2-electrode

Temp

Pt-100

connection of Pt-100

Gasmix

Socket, 15 pin

connection of a Gasmix-unit

Host

Socket, 9 pin, RS485 or RS422

connection of a host computer

Recorder

Socket, 15 pin

connection of a recorder

Printer

Plug, 9 pin

connection of a printer

Ext. Signals

Socket, 15 pin

connection of remote signals

Substrate

Plug, 9 pin

connection of external substrate pump

Table 2-1: Dimensions and connections of the BIOSTAT® B BAE_B Rev. 2.31 - 1099 SW 4.1

2-4

2

Operating Manual BIOSTAT B

Delivery and Installation

2.5

Connection Specifications

2.5.1

Connectors, tubings, cables and Fittings




The connectors, tubings, cables and fittings required for laboratory connection and assembly of the BIOSTAT® B are included in the delivery, unless otherwise agreed in the order (i.e. for customized configurations). Details about the dimensions and specifactions are given below.




However the delivered parts may differ from the specifications below, if necessary for a customized configuration or for connection of additional peripheral devices. If questions arise concerning the applicability of the parts for a specific purpose, please contact your B. Braun Biotech sales representative or B. Braun Biotech International GmbH directly.

1.

You must only use connectors and fittings which B. Braun Biotech International GmbH has released for use with the BIOSTAT® B and its peripheral devices or for which B. Braun Biotech International GmbH has confirmed the applicability in writing. This is especially true for replacement of connectors and fittings in case of damages or wear and tear.

2.

Allways verify that the supplies of energy, water and air (or gasses) are designed according to the requirements of the fermentor system. Cooling water and air (or gasses) must be clean and free of corrosion products from the supply lines. If necessary, install corresponding prefilters in the supplies. Connection

Fittings

Dimensions

Function, special notes

Mains supply

fixed cable / shock-proof plug

see below

Cooling water õ

hose connector PVC fabric hose

hoses ID = 12 mm cooling water supply to control 12.7 x 18.7 mm unit

Cooling water

hose connector PVC fabric hose

tubings, ID = 8 mm connection to cooling water re8 x 14 mm turn in the laboratory or to lab. drain

Air õ

hose connector

tubings, ID = 8 mm connection to air supply or other 8 x 14 mm gas supply systems;

O2 õ

hose connector

connection of extra O2 supply; combined with air inlet, if no extra gas supply should be used

at first installation the customer or our customer service has to attach the plug which corresponds with country standard

Table 2-2: Dimensions of the tubings and connectors 2.5.2

Mains Connection

There are 2 different versions available of the BIOSTAT® B which can be connected to the following voltages. Check on the rating plate of the BIOSTAT® B system delivered to you, whether you received the correct voltage version :


Cat. No. 884 032/6 with 230 V, 50/60 Hz




Cat. No. 884 033/4 with 115 V, 50/60 Hz

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2.5.3

Laboratory Connection for Cooling Water Supply and Return

2.5.3.1

General and Safety Information




The BIOSTAT® B requires a precontrolled laboratory water supply of max. 2 barg, without pressure deviations, see the above P&I diagram. The cooling water will supplied to the thermostate system and to the exhaust cooler. For details about a thermostate system of customized design see the P&I diagram supplied for your fermentor system.




The water must be clean and free of particles. A dirt filter is to be installed either in the laboratory installation or in the feeding line to the fermentor control unit.




The minimum operating temperature in the culture vessel will be about 8 degC above the cooling water temperature. For operation at a lower temperature you will need an external cooling device.




The water hardness should be below 12 German Degrees of Hardness in order to prevent calcareous deposits. Note the conversion table for degrees of hardness in the supplement.




Always verify the correct connection of the cooling water supply and drain/return lines to their inlet and outlet connections at the control unit. The female and male quick connectors are especially assigned and labelled correspondingly, so that they cannot be mixed up.




If the BIOSTAT® B is moved to another working place and the utility connections are rebuilt, take special care to properly connect the supply and the return/drain lines to their corresponding in- and outlets. Erroneously connection of the cooling water supply to the “Cooling water outlet “ connector of the control unit can result in supplying overpressure to the culture vessel, which may cause the vessel to burst.




BIOSTAT® B, cat-no. 8840326 (230 V, 50/60 Hz) of unit no. 3226 and later, as well as cat.-no. 8840334 (115 V, 50/60 Hz) of unit no. 3216 and later, have a return valve in the cooling water outlet installation of the control unit. This prevents supplying overpressure to the culture vessel, if the cooling water supply is erroneously connected to the cooling water outlet.




Elder versions of the BIOSTAT® B can be rebuilt with the „Rebuilding kit return valve heater BIOSTAT® B“, cat.-no. 34108971. Please contact your representative of B. Braun Biotech International GmbH, if you want to have your BIOSTAT® B rebuilt. Rebuilding must be done by well trained and qualified service personnel.




The cooling water outlet / drain / return line must not be blocked. The temperature circuit of control unit and culture vessel must always operate at ambient pressure. Overpressure must not be supplied to the culture vessel jacket since the glass vessel has a limitted pressure resistance. Overpressure can cause the vessel to burst. This is also true if the BIOSTAT® B is connected to an external cooling device or a closed cooling circuit in the laboratory.

2.5.3.2

Connection of the Cooling Water Supply and Drain/Return

1.

Connect the laboratory cooling water supply to the „Cooling Water Inlet õ“ located at the left side of the control unit, with the tubing included in the delivery set. – connection: 2 bar (29 psig) controlled, – required flow: max. 5 l/min.

2.

Connect the „Cooling Water Outlet “ located at the left side of the control unit to the laboratory drain, waste water line or the return connection of a closed laboratory cooling water installation. The required tubing is included in the delivery set.

3.

When a free drain is applied in the laboratory, place the outlet line in such a way that it descends by a steady gradient, in order to avoid water pockets in the line.

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2.5.4

Connection of Air or Gas Supply




The BIOSTAT® B can be connected to any pre-controlled pressurized air supplies delivering max. 0.8 barg (approx. 11.6 psig). For O2-enrichment an additional source supplying a gas enriched with oxygen can be connected. For details see the P&I diagram.




Optionally a Gasmix unit is available for supply of a mixture of 4 gases.




The air or gas (mixture) fed into the system must be dry and free from oil and dust. If necessary, prefilters must be installed on the laboratory side.




Defects and damages caused by contaminated air or gas will not be covered by our warranty.




In the inlet tubings to the culture vessel and for the exhaust membrane filters provide sterile gas supply (filter no. 1.2) and exhaust (filter no. 1.4) of the culture vessel.

1.

Connect the laboratory air supply to the „Air Inlet“ which is located at the left side of the control unit, with the tubing included in the delivery set.

2.

For O2 - enrichment connect an extra oxygen source to the O2 inlet connector. If no extra oxygen supply should be connected, the O2 inlet is combined with the air inlet by an adapter.




If the laboratory supply delivers a higher pressure, you must install a pressure reducer to preadjust the pressure.

3.

The air inlet tubing of the culture vessel and the exhaust equipment will be prepared and connected while preparing the fermentation run. See section 3 and 4 for details.

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STARTING AND OPERATING THE BIOSTAT® B

3.1

Preparing and Connection of the Control Unit

3.1.1

Preparing the Workplace

1.

Place the control unit, the culture vessel and the peripheral devices together with all accessories at the working place. Verify, that there is enough space for convenient handling.

2.

Set up the control unit at the operating site and connect it to the laboratory installations, see chapter 2 for details. : – the power supply, – the cooling water supply and waste water drain, – if required, an external thermostate system, – and the air supply (if required, together with the extra oxygen supply)

3.1.2

Connecting the Thermostate System




If the BIOSTAT® B has been moved to another working place and the cooling water supply and drain/return line has been rebuilt, verify that the inlet and outlet connectors are properly connected. See the installation instructions in section 2 for details.

1.

Check the connection of the laboratory cooling water supply to the inlet “Cooling water “ of the control unit and the connection of the laboratory drain or cooling water return line to the outlet “Cooling water “. Connect the tubings supplied with the system, if not yet done so. Verify that the laboratory supplies provide cooling water at 2 barg max. and 5 l/min.

2.

Check connection of the cooling water outlet to the laboratory drain or cooling system. When using a free drain, the hose must descend steadily in order to avoid water pockets.




If an external cooler or a closed cooling system is connected, verify that the thermostate system can operate at ambient pressure and cooling water is circulating sufficiently in order to maintain the desired temperature in the culture vessel.

3.1.3


3.1.4

Connecting the Air/Gas Supply System Check the connection of the laboratory air/gas supply at the air inlet at the left side of the control unit and connect, if necessary. The laboratory supplies must deliver about 10 l/min of air (or gas) at 0.8 barg (11.6 psig). Preparing and Connection of Peripheral Equipment for Measurement and Control

1.

For checking and calibration of the pH-electrode, which must be done before it is mounted into the culture vessel, you can connect the electrode to the corresponding socket at the side panel of the control unit. For details about calibration see section 4.

2.

For checking the pO2-electrode, e.g. after service (replacement of the membrane, etc.) you'll have to connect the electrode to its amplifier and polarize. For details see section 4.

3.

For the start of the fermentation run the electrodes will be connected to the corresponding sockets at the control unit after autoclaving, when the culture vessel is prepared and connected for the fermentation run.

4.

As far as necessary connect the cables of an external recorder, printer, a gas mixing system or a host computer, respectively. See the corresponding plugs and sockets on the left side of the control unit.

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3.2

Preparing the Culture Vessels and Accessories

3.2.1

Checking the Equipment




During assembly of the culture vessel and mounting of the probes and accessories, carefully check all parts for damages. You should examine particularly closely the glass vessel itself, the seals on the top plate and the O-rings of all mountings. See the instructions in Chapter 4 concerning the testing and maintenance of the vessels.

Warnings:


Damaged glass vessels may no longer have the bursting strength required for autoclaving or running the fermentor, respectively, and must be replaced immediately.




Damaged seals can be the cause of contaminations of the culture during the process. Seals on accessories, which are often refitted, should be replaced regularly.

3.2.2

Assembling and Equipping the Culture Vessels




The following sections include a summary of the required or possible steps. The handling of the different culture vessels and their equipment is described in details in section 4.

1.

Unscrew the top-plate screws and dismount the top-plate (1). Take care not to damage the O-ring (9) and clean, if dirty or replace, if damaged.

2.

Remove, replace or mount, respectively, any internal accessory required for the intended process. Mount all parts which are to be installed into their top-plate port from below. Any port where no accessory part is installed, must be fitted with a blind closure.

3.

Check all seals carefully and replace any seal which appears to be defective. You may grease seals slightly with silicone grease to prevent glueing onto their contact surfaces.

5.

Mount the top-plate back to the glass vessel. Locate the top-plate with the knurled screws. Tighten the screws properly handtight. Do not use a spanner, you may damage the threads and/or the glass vessel. Tighten crosswise to prevent uneven pressure of the top-plate against the glass flange of the vessel and the sealing.




Uneven pressure applied to the rim of the glass vessel can damage the vessel.

6.

Complete equipment of the top-plate with all probes and accessories of the delivery set required for the fermentation run.




The pH-electrode has to be calibrated before it is installed. The calibration of the pO2 - probe will be carried out in oxygen-free culture medium after autoclaving.

7.

Arrange the electrodes and accessories on the top-plate from the inside to the outside so that they are easily accessible. For the parts and suggestions of their placement in the top plate see fig. „Top plate of a BIOSTAT® B culture vessel with recommended placement of the accessories“ and the corresponding information in section 4.

8.

Attach a short piece of silicone tubing (4x7 mm) to one side of the membrane filter for the air inlet and the other end to the hose connector of the sparger pipe in the top plate. Place the tubing into the clamping device on the culture vessel so that it is completely clamped off and no culture solution can enter the air inlet line via the sparger ring.

9.

Screw the exhaust cooler directly or with its flexible connector into its port in the top plate. Connect the membrane filter for the exhaust to the exhaust cooler via a short piece of silicone tubing of 4x7 mm.

10.

Adjust the height of the antifoam electrode and the level electrode according to the planned working volume. The height can be determined by gauging the capacity by liters.

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11.

Wrap aluminum foil round exposed electrical connectors and clamp off all tubings of those parts which reach into the culture solution (such as inlet air tubing, harvest pipe, for instance). The air/gas supply line can be clamped off by means of a clamping device („dovetail“) and fixed to the vessel.




The exhaust line, must be kept open so that pressure compensation with ambient pressure can take place, as it is necessary during and after the sterilization in the autoclave.

12.

Connect all the necessary peripheral equipment (storage bottles for corrective solutions, sampling systems etc.). For equipment and preparation of the storage bottles (for acid, alkaline solution, antifoam agent) with withdrawal pipes, silicone tubes and exhaust filters and filling the bottles) see corresponding sections below.

3.2.3

Filling the Jacket of the Culture Vessel

Note: The thermostate liquid in the jacket of the culture vessel provides optimum heat transfer during sterilization and in the process. Therefore, before sterilizing the culture vessel and prior to any new fermentation run, it is necessary to check the filling of the jacket and refill with thermostate liquid, if necessary. For this you can proceed as follows: 1.

Connect the thermostate tubings to the culture vessel in this way: – connect outlet at supply unit to lower quick connector at culture vessel jacket (inlet) – connect upper connector at culture vessel jacket (outlet) to inlet of supply unit. The connectors are marked correspondingly and cannot be mixed.

2.

Switch on the basic unit (MAINS 0 -> I). Press FILL THERMOSTATE switch to fill the thermostate system and the jacket of the culture vessel. After filling the jacket disconnect again the thermostate hoses and continue preparing the culture vessel for sterilization.




The quick coupling at the lower inlet keeps the jacket inlet closed. The tubing connector at the upper outlet is open to provide pressure compensation with ambient air.

Caution: Do not close (or clamp off) the upper outlet. The thermostate liquid expands during heating-up in the autoclave. Excess liquid must be allowed to flow out of the jacket. Else overpressure will be formed in the jacket which may destroy the vessel. 3.2.4

Filling the Culture Vessel

1.

Open any port in the top plate and introduce the culture solution. Please note that the culture solution partly evaporates during sterilization. The loss amounts to approx. 100 ml per 30 min sterilization time (the exact which loss can only be ascertained empirically).

2.

Allow for this by increasing the quantity of culture solution in the vessel before sterilization or by subsequently introducing additional solution.

3.

If the culture medium required for later fermentation is not autoclavable, fill in water instead to ensure reliable sterilization of the culture vessel (volume to be added should correspond to the loss through evaporation; recommended volume is approx. 200-300 ml).

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3.2.5

Preparing and Connecting Addition Agent Bottles

The delivery set of the culture vessels includes 3 bottles for corrective solutions (acid, alkaline, antifoam or nutrient solution). These are 250 ml bottles for the vessels B 2 or B°2 - airlift and 500 ml bottles for the vessels B 5, B°5 - airlift and B 10. These volumes usually will suffice for 1 process. However, especially for supply of nutrient solutions and during long-term fermentations or continuous operation we recommend to prepare several bottles in order to have sufficient volumes available. 1.

Fill the bottles with acid, alkaline, antifoam or nutrient solutions as required.




Caution: When using acid or alkaline agents take precautions against burns. Use protective gloves, for instance.

2.

Attach a piece of silicone tubing to the hose connector carrying the rising pipe of the bottle.

3.

Connect the other end of the silicone tubing to one of the nozzles welded in the culture vessel top-plate. The tubings should have sufficient length to allow for passing through the peristaltic pump in the control unit.

4.

Place the bottles into the holders on the frame supporting the culture vessel. Clamp-off the tubings before autoclaving so that no corrective solution can escape from the bottles. For this you can use the clamping device („multible dovetail“) attached to the culture vessel.

3.2.6

Sterilization of the Culture Vessel

1.

Take care that all open ports in the top plate of the culture vessel are closed. Locate all loose parts so that they cannot get loose during handling and transport.

2.

Cover the electrode plugs with some aluminium foil in order to protect them from the impact of steam. Some electrodes (pH-, pO2-electrode) may have special protective caps for this included in the delivery.

3.

Place the bottles for corrective solutions into their holders at the culture vessel. The holders will facilitate transport of the vessel and the assemblies and save space in the autoclave.

4.

Locate the tubings of the air / gas inlet and of the corrective solution supplies. For this you can use the clamp devices (dovetail clamps) attached to the culture vessel.




The tubing of the exhaust filter must not be clamped-off. This is necessary to provide the pressure compensation required during and after the sterilization.

5.

The double jacket of the culture vessel must be completely filled with the thermostate liquid (water). If the vessel is used for the first time or to add missing liquid, connect the in-/outlet to the control unit and push the FILL THERMOSTATE button to fill the double jacket.

6.

Autoclave the culture vessel and all accessories at a temperature of 121 degC.




The temperature in the centre of the culture vessel must be kept at 121 degC for at least 20 min. If necessary, test the reliability of your autoclave in preliminary tests by sterilizing suitable indicators (commercially available test spores, for instance).




Proper sterilization requires approx. 40 min for the 2 l-culture vessel and approx. 60 min for the 5 l-vessel. Please check whether these times are sufficient for your individual setup. For the 10 l vessel the time has to be estimated empirically.

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3.3

Peparing a Fermentation Run

3.3.1

Overview

Note: This overview should help experienced users to check the necessary steps of operation. For a detailed description of the individual steps see the sections 3.4.2.ff below. 1.

After sterilization you should let the culture vessel and the connected equipment in the autoclave cool down. Before removing the equipment from the autoclave, check whether the parts are still hot. Put on protective gloves, if necessary.

2.

Now the culture vessel can be brought to the workplace for further set up and connection. Take special care during transport to prevent loosening of tubings or assemblies connected.

3.

Mount the motor onto the stirrer shaft coupling at the culture vessel.

4.

Connect the gas/air supply to the inlet gas/air filter.

5.

Connect the thermostate system to the culture vessel.

6.

Connect the cooling water supply and backflow tubing to the exhaust cooler.

7.

Remove protective aluminium foil from the electrode connectors. Connect the cables to the electrodes and to the corresponding sockets at the control unit.

8.

Switch on the control unit. If necessary compensate again for losses of thermostate liquid in the jacket of the culture vessel.

9.

Place the tubings of the additive agent supplies into their corresponding peristaltic pumps and fill the hose lines manually to compensate for the clearance volume in the tubings.

10.

Check display of the operation parameters at the measurement and control system and adjust as far as necessary. See below and section 5 or this manual for details.

11.

Inoculate thevessel with the seed culture. See below for details about suitable methods.

3.3.2

Final Setting-up and Connection of the Culture Vessel for Fermentation

3.3.2.1

Connecting the Thermostate System

1.

Connect tubings of the thermostate system. Check whether the water supplied from the laboratory has a controlled pressure of 2 bar (29 psi). Readjust before opening the supply.

2.

If necessary compensate for possible losses of cooling water in the vessel jacket (see „Filling the vessel for the first time“). Excessive water will escape from the outlet port.




During the fermentation run cooling water will automatically be introduced into the thermostate system when the temperature in the culture vessel exceeds the setpoint temperature.

3.3.2.2

Connecting Air / Gas supplies

Note: The pO2-electrode must be calibrated before starting the air supply. Note the more detailed information about pO2-calibration in corresponding chapters of section 4 and 5 below. -> 3.3.2.3

Remove the air supply tubing from its holder on the vessel, then fit the filter into the holder. Connect the hose to the hose connector marked „AIR out“ on left side of the control unit. Connecting the Exhaust Cooler

->

Connect the cooling water supply and drain to the exhaust cooler. See the fig. in section 4 for the location the connectors.

❏

There will be a constant flow of cooling water through the exhaust cooler according to the flowrate adjusted in the laboratory connection.

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3.3.2.4

Fig. 3-1:

3.3.2.5

Connection of Corrective Solution Supplies

Pump head of the peristaltic pump; ❏ insertion of tubings and adjustment of pressuring load; ❏ the pump is switched-off with manual switch in central position !

1.

Place the tubings of the bottles containing into the corresponding peristaltic pumps in the control unit :

2.

Lift the lid of the peristaltic pump and open the inlet/outlet clamps. Place the tubing into the rotor from the inlet side (lower clamp). Turn the rotor while inserting the tubing. Fasten the tubing with the clamps.

3.

Check the pressure load of pump head rolls and readjust, if necessary. They should fully clamp the tubing and prevent any backflow of liquid, but must not damage the tubings.

4.

You may turn the pump head by hand until the adjustment screws are accessible. Turn the screws clockwise to increase the pressuring load and vice versa to reduce. Finally close the lid.

5.

Switch on the pump manually to fill the tubing with the liquid up to its end. This will prevent incorrect dosing and calculation of the delivered volumes.

1.

The motor is connected to the control unit by a fixed cable, which is internally wired in the control unit. It is ready for use after mounting on to the stirrer shaft of the culture vessel.

2.

Place the motor onto the adapter on the stirrer shaft of the culture vessel. The support of the motor fits into the locating pin at the adapter.

Connecting the Drive Motor

See fig. on left side for correct assembly of the motor.

Fig. 3-2: Connection of the drive motor

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3.3.3

Connecting the Probes and Setting the Measurement and Control System

3.3.3.1

Overview on Connection and Preadjustment

1.

If not done yet, connect the electrode cables to the electrodes and to the corresponding input sockets on upper left side of the control unit.

2.

Switch on the control unit and check that the equipment is functioning correctly. For this observe the display in the operating terminal for error messages.

3.

Select required functions and adjust the necessary parameters at the measurement and control system. See fig. and overview on the necessary steps below.

Note: For detailed information about the measurement and control system of the BIOSTAT® B, such as display functions, necessary adjustments of measurement and control parameters or error messages, for instance, see section 5 of this manual. 3.3.3.2

Steps of Operation of the Measurement and Control System

1.

Select main function with main function key. Changing to the required main function is possible from any menu or submenu selected.

2.

Select required menu/submenu of the main function by repeatedly pressing the main function key until the required menu is displayed.

3.

For entering data move on to the required position with „Cursor up/down“. Enter numerical data via the numerical keypad. For entering non-numerical inputs select required input with „ALTER“nate-key.

3.3.3.3

Preadjustment of Operating Parameters and Actuators

1.

Calibrate the pO2 electrode in the culture vessel. See more detailed information about calibration in section 5 or in the documentation delivered with the electrode.

2.

Select the required air flow rate on the rotameter on the front panel of the control unit.

3.

Enter the required setpoints for the measurement and control parameters, for instance : – Desired operating temperature – Stirrer speed – Minimum and maximum pH values of pH controller – pO2 limits and mode for controlling oxygen supply – Response threshold of antifoam probe – Response threshold of level probe

3.3.3.4

Preadjustment of the Supply of Corrective Solutions




In order to avoid incorrect calculation of dosages or dosing times due to the volume of the connecting hoses, these must be filled with the necessary corrective solutions before proceeding to fermentation.

1.

If not yet done with the connection of the tubings to the peristaltic pumps, now press again the MAN - switch on the corresponding pump module in the control unit.

2.

Allow the pumps to run until the tubings are filled with the corrective solution right up to the inlet nozzle at the culture vessel.

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3 Operating Information

3.4

Carrying-Out a Fermentation Run

3.4.1

Sterility Test

Damaged parts, especially faulty seals, but also incorrect handling while equipping the culture vessels, can result in contaminations of the culture medium. This can cause the process to fail. 1.

In order to detect evidence of incorrect handling or faults, especially concerning seals, you may carry out a sterility test before starting the fermentation run.




Conditions: all necessary components should be connected to the culture vessel; the necessary operating conditions (temperature, air/gas supply etc.) should be set.

2.

You can carry out this sterilization test very simply by starting the test run at the same time you start the inoculation culture and allowing the fermentor to run until the inoculation culture is finished.

3.

A 24 h test period will detect most infections by common environmental germs. It should leave you enough time to check, reassemble and prepare the culture vessel again for the planned fermentation run, if infections occur.

3.4.2
3.4.2.1

Inoculating the Culture Vessel Inject the inocculation culture. There are various inoculation techniques among which you may chose. You will find detailed information on individual procedures below. Inoculating the Using a Syringe

1.

Remove the necessary amount of solution from the inoculation culture with a syringe and transfer it to the culture vessel using suitable measures to avoid contamination.

2.

Remove the blind closure from an inoculation port equipped with a septum membrane. Flame the port with an open flame (e.g. using a bunsen burner) to prevent contamination of the membran. You may also wet the membrane with disinfectant and allow it to take effect for approx. 5 min.

Note: Using disinfectants may be less effective. Some of the disinfectant can be introduced into the vessel while the syringe is pierced through the membrane and then interfere with culture growth. It may also contain resistant spores which will then contaminate the culture. 3. 3.4.2.2

Fit the syringe with a sterile needle and puncture the membrane in order to transfer the inoculation solution to the culture vessel. Inoculating the Culture Vessel with a STT Quick Connector




B. Braun Biotech International GmbH offers a STT quick connector, which provides easy and convenient sterile access to the culture vessel for supply of additional solutions, seed cultures, etc..




Refer to section 4 for information about use and connection of the STT-quick connector. Detailed information are given in the „Operating Information STT Quick Connector“, which is delivered with this device

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3.4.3

Adding Sterile Solutions during the Fermentation Run

3.4.3.1

General Notes

In certain fermentation processes, it is often necessary to introduce additional nutrient solutions or special essential nutrients (vitamins, amino acids etc.). Note: Solutions must be transferred from the sterilized storage bottle and to the culture vessel while avoiding any contamination of the culture.
3.4.3.2

Add doses of additional substances under sterile conditions. There are various techniques among which you may chose. Further information on individual procedures is given below. Transferring Solutions by Means of a Syringe

1.

Suck sterile solution into syringe from storage bottle, etc. When using solutions which cannot be sterilized by heat, use a sterile needle connected with a sterile filter.

2.

Proceed as shown above for inoculation using a syringe.

3.4.3.3

Transferring Solutions with a STT Quick Coupling

1.

Refer to the „Operating Information STT Quick Connector“, which is delivered with this device, for information about the use and handling of this device.

2.

Transfer the sterile solution to the culture vessel using the force of gravity or by means of a peristaltic pump.

Note: The female connector can be utilized several times; different male connectors can be inserted one by one in order to transfer various solutions. 3.4.4


3.4.4.1

Taking Samples Samples will be taken from the vessel when required. There are various sampling techniques among which you may chose. A standard method (use of the harvest pipe) is shown below. For other suitable methods see section 4. Taking Samples Using the Harvest Pipe

Samples can be removed from the culture vessel via the harvest pipe integrated into the top plate and to which a sterilized silicone hose clamped off with a hose clamp is connected. 1.

Insert the connecting hose into an external receptacle (a bottle or beaker, for instance).

2.

Open the hose clamp. The overpressure caused by aeration of the vessel usually suffices to transfer the sample to the receptacle.




When using high-viscosity culture broths, you can clamp off the exhaust hose for a short time in order to raise the inner pressure forcing out the sample.

3.

Dip the open end of the hose into disinfectant between two samples, in order to avoid a contamination.

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3.5

End of a Fermentation Run

3.5.1

Harvesting the Product

1.

Remove the product and the culture medium containing the cells as required for their further use. Possible methods are, for instance: – Removal via the harvest pipe and through a transfer tubing for use as seed culture (transfer to a larger culture vessel for scaling-up). – Removal via the harvest pipe under sterile conditions (under a Laminar-Flow - chamber) and filling into sterile harvesting bottles. – Sterile or insterile downstream processing of the medium.




Take note of the risks of your application and follow the related safety precautions and other legal or elsewhere compulsory regulations to avoid personnel hazards.

2.

Except for processes where microorganisms are applied, which are „generally regarded as safe“ we recommend to fill the culture vessel with water and sterilize again in the autoclave before disassembling for cleaning, maintenance and rebuilding for the next use.

3.5.2

Cleaning and Maintenance

Cleaning or maintenance intervals in general depend on how fast the culture vessel and the equipment gets dirty (due to sticking of proteins, fouling, etc.). 3.5.2.1

Zwischenreinigung nach Fermentationen




For intermediate cleaning between two fermentation runs it often suffices to rinse the culture vessel thoroughly with water.




When the system will not be used for a short time it is advisable to fill the vessel with demineralized water. It is not necessary to remove electrodes and accessories for this. The water protects the electrodes from drying out.

3.5.2.2

Basic Cleaning




The culture vessel and the glass bottles for corrective solution supply can be cleaned in a laboratory dish washer. For this the glass culture vessel need to be removed from the stainless steel supporting frame and the top-plate with all accessories need to be dismounted. Refer to section 4 for dismounting the culture vessel.




When the culture vessel is contaminated with organic substances you can clean the glass surface with the special laboratory glass cleaners available commercially (e.g. RBS from ROTH, NEODISHER etc.) and which are dissolved in warm water.




Anorganic deposits can be dissolved using diluted hydrochlorid acid or a similar agent. Take care to protect yourself against burns. When using aggressive cleaning liquids carefully flush the vessel with water afterwards.




The metall parts (top-plate, etc.) can be cleaned mechanically or using alcohol or commonly used detergents. Also flush the vessel carefully with water afterwards.




O-rings, seals and washers can be cleaned mechanically (chemical detergents may affect the rubber material, for instance). However you should take special care, not to damage the seals. You may grease the seals with some silicone grease. Above this, it is recommended to replace all seals regularly.

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4

EQUIPMENT OF THE CULTURE VESSELS

4.1

General Information

4.1.1

Delivery and Starting up

All BIOSTAT® B - fermentors undergo a comprehensive final quality check before the delivery. This includes all components and accessories as ordered for the fermentor system. In addition to this, a sterility test with a duration of 100 h is carried out on the system. The culture vessels will usually be delivered premounted with the component parts of the standard equipment. The electrodes, additional accessories and peripheral equipment included in the order will be packed separately and can be mounted and connected as required for the intended process. The section 4 below includes the information about the culture vessels available for the BIOSTAT® B and their standard equipment. If the BIOSTAT® B is to be used for special cultivation processes, such as tissue cell cultivation, for instance, it may be necessary to remove or replace existing equipment or to add additional parts. Section 4 also included the information about commonly used additional equipment for general use or as applied for specific culture vessels.


If your fermentor is of customized design or has component parts not shown in section 4 you should refer to section 7 of the manual. Section 7 is reserved for customized vessels, customized equipment and optional extensions not commonly used for the BIOSTAT® B.




You will get a corresponding documentation update with the Operating Manual or by extra mail. Please contact your B. Braun Biotech representative or B. Braun Biotech International GmbH, if no documentation is delivered for a customized modification of the BIOSTAT® B.

4.1.2

Inspecting a Culture Vessel and its Accessories

For the first setup after delivery and prior to any fermentation run we recommend these measures:


Check whether the equipment is complete, as required for the process – for internal and external equipment of the culture vessels – for connection of the culture vessel to peripherals and to the control unit




Handle the culture vessels and component parts with special care and check whether they are in good condition:

1.

Carefully inspect the glass culture vessels and other equipment made of glass. These parts must be free of damages.




The culture vessel can be subject to pressure during the sterilization. Damages will reduce its pressure resistance and thus its bursting strength.

2.

Inspect all ports in the top plate as well as all electrodes and accessories which should be fitted into these ports including their seals and O-rings. Make sure that all contact surfaces are free from adhesive deposits. Clean carefully, if necessary. Replace seals and O-rings at the slightest suspicion of a damage (when pressure marks, are visible, for instance).

3.

We recommend to slightly lubricate the seals and O-rings and their contact surfaces with silicone grease before fitting. This can usually prevent sticking of the seals due to the impact of high temperatures during autoclaving, and thus can curb premature wear.

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Equipment of the culture vessels

4.2

Culture Vessels

4.2.1

Dimensions and Design Features of the Culture Vessels

Culture vessel

B2

B5

B 10

B2 - Airlift

B5 - Airlift

total volume [l]

3

6,6

13

3,5

9

working volume [l]

2

5

10

2

5

1,6:1

2,2:1

2,5 :1

5,4:1

5:1

4:1

3,7:1

H/D-ratio (total) H/D-ratio (work. vol.) space requirements ca. (HM,B x D [mm])

587 x 350 incl. motor

692 x 400 incl. motor

828 x 420 incl. motor

692 x 350

850 x 420

autoklave dimensions - for standard cooler - cooler bent over (H x D) [mm]

550 x 350 380 x 350

700 x 400 485 x 400

820 x 420 610 x 420

740 x 350 565 x 350

900 x 420 690 x 420

height incl. exh. cooler (HK, [mm])

508

645

767

692

850

vessel height with top plate (HG, [mm])

285

390

512

469

595

height of glass vessel (HGl, [mm])

240

345

470

430

553

inner Ø vessel (Di,Gl, [mm])

130

160

190

130 (head room) 80 (reactor)

190 (headroom) 110 (reactor)

4 x Ø 19 2 x Ø 12 9xØ6 4 hose connectors

4 x Ø 19 1 x Ø 12 6xØ6 4 hose connectors

3 x Ø 19 2 x Ø 12 10 x Ø 6 4 hose connectors

top plate ports, number x Ø [mm]

2 x Ø 19 2 x Ø 19 2 x Ø 12 2 x Ø 12 9xØ6 9xØ6 4 hose 4 hose connectors connectors

side entry ports

4

4

no

no

no

top plate locating screws

3

6

6

3

6




You should compare these data with the equipment delivered to you. However, you should note, that B. Braun Biotech International GmbH reserves the right to change these specifications without prior notices.




Even for modified culture vessels the possible equipment and required handling will usually be the same as shown below. For a modified design and equipment, check whether additional information is added to section 7 of this manual or delivered extra. If you require additional information about specific component parts, you should contact your representative of B. Braun Biotech or B. Braun Biotech International GmbH directly.

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1

2 3

Di,Gl

4

DR

HM,B HM,M HR

HK HG

5 6

HGl,i VFl

Fig. 4 - 1:

Setup and dimensions of the standard culture vessels type B2, B5 and B10 with stirrer drive system (1) motor (2) exhaust cooler (3) reference thermometer (4) side entry hose connector (5) baffle insert (6) corrective solution bottles

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Equipment of the culture vessels

Fig. 4 - 2:

Setup and dimensions of a culture vessel type B2 - Airlift

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Equipment of the culture vessels

Fig. 4 - 3:

Setup and dimensions of a culture vessel type B5 - Airlift

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Equipment of the culture vessels

4.2.2

Set-up, Equipment and Accessories of the Culture Vessels




The tables below give an overview of the accessories available at present and described in this manual. More detailed information about the individual component parts is shown in the following chapters of section 4.




You should note, that the equipment of the vessels and the available accessories are subject to a continuos development and may be modified, added or replaced accordingly. Thus these information are not necessarily complete. For customized equipment you may refer to section 7 of this manual or to the documentation delivered extra. If the information given in this manual and the design and handling of your equipment do not match, please contact your representative of B. Braun Biotech or B. Braun Biotech International GmbH directly.

4.2.2.1

Basic Equipment of the Culture Vessels

Culture vessel

B2

B5

B 10

vessel insert

baffle insert with 4 baffles

baffle insert with 4 baffles

baffles

stirrers

3 x 6-bladed 2 x 6-bladed 2 x 6-bladed disc impellers; disc impellers; disc impellers; paddle stirrers paddle stirrers paddle stirrers optional optional optional

air / gas supply

pipe with ring sparger, silicone membrane insert optional

pipe with ring sparger, silicone membrane insert optional

exhaust cooler type

B2

temperature sensor

M2/B2 - Airlift M5/B5 - Airlift guiding tube (loop reactor)

guiding tube (loop reactor)

--

--

pipe with ring sparger, silicone membrane insert optional

pipe with ring sparger

pipe with ring sparger

B5

B5

B2

B5

Pt-100-200/4

Pt-100-300/4

Pt-100-400/4

Pt-100-300/4

Pt-100-300/4

pH-electrode

Pa/12 - 200

Pa/12 - 325

Pa/12 - 325; or as option: Pa/12 - 425

Pa/12 - 200

Pa/12 - 325

pO2-electrode

12/220

12/320

12/320; (12/420 option)

12/220

12/320

antifoam probe, insertion depth

50 mm

50 mm

std. 50 mm opt. 260 mm

50 mm

50 mm

122 mm

122 mm

122 mm

122 mm

level probe, insertion depth 3 storage bottles

std. 122 mm opt. 260 mm 250 ml

500 ml

500 ml

250 ml

500 ml

2 x Ø 19 2 x PG 13,5 9 x Ø 11

2 x Ø 19 2 x PG 13,5 9 x Ø 11

4 x Ø 19 2 x PG 13,5 9 x Ø 11

4 x Ø 19 1 x PG 13,5 6 x Ø 11

3 x Ø 19 2 x PG 13,5 10 x Ø 11

sampling system

x

x

x

x

x

manual sampler

x

x

x

x

x

blind closures

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Equipment of the culture vessels

4.2.2.2

Accessories for the Culture Vessels

Cat.-Nr.

Accessory

Description, use

3833697/9

hose connector

hose connector with Øo = 4 mm - assembly into 6 mm top-plate port

884 431/3

quadruple hose connector B

adapter with 4 hose connectors of Øo = 4 mm - assembly into 19 mm top-plate port

884 463/1

inoculation adapter B

septum kit with membrane and protective cap, - assembly into 6 mm top-plate port - to be punctured using a syringe or small needle; - self-sealing membrane which can be punctured several times

884 060/1

inoculation adapter B10

septum kit with membrane and blind closure - assembly into 19 mm top-plate port - for 1-channel inculation kit, cat.-no. 884061/0

884 061/0

1-channel inculation kit

inculation kit für inculation port B10, - hollow needle of Øo = 6 mm - delivered with sterile sleeve

3833717/7

reduction adapter 19/6

adapter for mounting of parts with Øa = 6 mm into top-plate ports of Ø = 19 mm

881 007/9

filters for air inlet and exhaust

autoclaveable membrane filters in plastic housing - filter membrane of 0,2 µm - with hose connectors

3922830/4

filter element for exhaust B 10

plastic filter tube with membrane filter - filter membrane of 0,2 µm

884 459/3

flexible holder for exhaust cooler

piece of tubing for exhaust cooler assembly, can be bent over for autoclaving, requiring a lower inner height of the autoclave

884 432/1 884 453/4

control thermometer - for vessels B2 - for vessels B5 and B10

for visual monitoring of temperature, - insertion into pocket in top-plate port of 6 mm; - removable for autoclave

880924/0 880941/0

STT quick connector (female) - for tubings 3.2 x 1.6 mm - for tubings 1.6 x 1.6 mm

female connector of STT connector for sterile connection of external supplies, to be installed to the tubing attached to the culture vessel

880920/8 880940/2

STT quick connector (male) - for tubings 3.2 x 1.6 mm - for tubings 1.6 x 1.6 mm

male connector of STT connector for sterile connection of external supplies, to be installed to the tubing attached to the external supplies

Bypass sampler kit M/B - for tubings 1.6 x 1.6 mm

for setup of a bypass line for removal of small samples (or for introducing small volumes of corrective solution); - membrane holder and septum membrane - 2-channel connector for top-plate port of 19 mm - 2 m of silicone tubing 1.6 x 1.6 mm - 0,2 m PTFE hose 3.5 x 5.5 mm

884 434/8

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Equipment of the culture vessels

Cat.-Nr.

Accessory

Description, use

884438/0 884457/7 884050/4

Harvest filter - for culture vessel B2 - for culture vessel B5 - for culture vessel B10

for removal of cell-free samples out of cultures with microcarrier-bounded cells - assembly into 6 mm top-plate port

884466/6 884467/4 884048/2

Silicone membrane insert - for culture vessel B2 - for culture vessel B5 - for culture vessel B10

vessel insert carrying a silicone membrane wrapped around, which is used for bubble-free gas supply to the culture mediam (especially applied for tissue cell cultures); kit delivered with accesories of gas supply and paddle stirrers

3823907/8 3823954/0 884054/7

Paddle stirrers - for culture vessel M2/B2 - for culture vessel M5/B5 - for culture vessel M10/B10

for optimum mixing of the medium at low stirrer speed

880830/9 880831/7 880832/5

Spinfilter - type B 2, 20 µm - type B 2, 40 µm - type B 2, 75 µm

vessel insert for separation of cell-free medium for sampling and harvesting; for use together with the short harvest pipe SF, cat.-no. 880826/0

880833/3 880834/1 880835/0

- type B 5, 20 µm - type B 5, 40 µm - type B 5, 75 µm

for use together with the short harvest pipe SF, cat.-no. 880827/9

884055/5 884056/3 884057/1

- type B 10, 20 µm - type B 10, 40 µm - type B 10, 75 µm

for use together with the short harvest pipe, cat.-no. 880859/8

880 826/0 880 827/9 884 059/8

Harvest pipe SF - for spinfilter B 2 - for spinfilter B 5 - for spinfilter B 10

especially designed harvest pipes for removal media (filtrate) separated by a spinfilter - assembly into 6 mm top-plate port

perfusion module - type B 2 - type B 5 - type B 10

modular perfusion kit continuous fermentation

884436/4 884439/9 884049/0

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Equipment of the culture vessels

4.2.3

Top plate Design and Placement of the Accessories

Fig. 4 - 4: Top plate Design and Placement of the Accessories - example for standard culture vessels type B2 and B5 port

recommended use

port

recommended use

N1

exhaust cooler

N 10

reserve

N2

Pt-100 temperature sensor

N 11

level probe

N3

antifoam probe

N 12

harvest pipe

N4

pO2 - electrode

N 13

pH - electrode

N5

supply of acid

N 14

air / gas inlet (sparger pipe)

N6

supply of alcaline agent

N 15

holder of baffle insert

N7

supply of antifoam agent

N 16

reserve

N8

supply of substrate

N 17

holder of silicone membrane insert

N9

reserve

Note: For the other standard culture vessels the arrangement of the ports is almost the same. Only their distance to the centre and the distance between the ports will be different.


For special and customized culture vessels the arrangement, type and number of top-plate ports can be different. Please check your equipment for this.

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Equipment of the culture vessels

4.3

Equipping and Assembling the Culture Vessels

4.3.1

Overview




The internal equipment of a vessel and the kind and number of components, which should be installed in the top-plate, need be planned carefully. Some of the accessory parts can only be mounted, removed or changed, while the top-plate is disassembled. Some parts are to be pushed through the top plate from below and screwed tight with a corresponding nut from above. Other parts will be tightened by corresponding nuts from below.

Fig. 4 - 5: Culture vessel type B 2; internal setup: (1) jacketed glass vessel (2) stirrer shaft (3) 6-bladed disc impeller (4) baffle insert (5) sparger pipe (6) sparger ring for air inlet (7) harvest pipe (8) antifoam probe (9) O-ring seal of top-plate

BAE_B Rev. 2.31 - 1099 SW 4.1




Parts for assembly while the top-plate is removed, are in general: – adapters for antifoam and level probe – adapters for the harvest pipe, – the adapter for the inoculation port – any universal fitting (hose connector) – the mounting pocket for the optional reference thermometer (if necessary) – tubing connectors for the side-entry ports of culture vessels B2 and B5




For culture vessels with drive shaft and stirrers such parts are, for instance: – baffle insert or extra baffles – the stirrers for the drive shaft – a spinfilter for media removal – a silicone membrane insert for buble free gas supply




For airlift culture vessels without drive such parts are, for instance: – guiding tube for loop system – electrodes, probes, pipes of special imension considering the height of the airlift vessels




Blind closures must be fitted to any topplate port, which is not required for equiment. For the blind closures of Ø = 6 mm, this is only possible, while the topplate is removed.




After assembly of the top plate to the glass vessel only these component parts can be added, removed or changed, which are located from outside: – exhaust cooler (or flexible adapter for exhaust cooler – antifoam and level probe – the harvest pipe – other component parts for assembly into clamping adapters – 2-way inlet of Bypass sampler – 4-way inlet adapter

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Operating Manual BIOSTAT B

Equipment of the culture vessels

4.3.2

Disassembling a Culture Vessel

1.

Disconnect the cables and tubings connected to the control unit. Remove the motor and all electrodes and component parts installed in the top-plate, as far as necessary for cleaning, maintenance and reequipment of the culture vessels.

2.

Turn the top-plate screws counterclockwise. They need only get loose from the flange of the stand, but need not be fully removed. Lift the top plate. Take care not to damage any seals if they are sticking. Take care not to damage any electrodes. Remove all parts which are not required for the next fermentation run.

3.

Clean the culture vessel inner and the component parts, if necessary. You need not remove the glass vessel from the stand for this. You may flush the parts with water. When the culture vessel is contaminated with organic substances you can clean the glass surface with the commercially availbale laboratory glass cleaners dissolved in warm water.




You may clean sticking residues of the culture medium mechanically. Make sure that the glass is not damaged. Anorganic deposits may be removed with a solution of hydrochloric acid or alike. When using aggressive agents rinse the vessel thoroughly afterwards.

4.

If the glass vessel is damaged and must be rebuilt, loosen the screws of the flange of the stand and remove the vessel. Insert the new glass vessel with special care. Do not hit the hose connectors of the culture vessel jacket, they can easily break.

5.

Check all parts for damages (especially the glass vessel and all seals/O-rings). If necessary (at sticking residues of the previous culture) clean the parts. Replace any seals and O-rings which are suspected to be damaged.




Order information for the glass vessels and specifications of the O-rings for the top-plate

4.3.3


for vessel type

B2

B5

B10

B2-Airlift

B5-Airlift

vessel cat.-no.

3920427/8

3920428/6

3920418/9

on request

on request

O-ring cat.-no.

39121992

39120155

3410006

39121992

3410006

specifications

136,12 x 3.53 EPDM

164,69 x 196,44 x 3,53 EPDM 3,53 EPDM

136,12 x 196,44 x 3,53 3.53 EPDM EPDM

Assembling a Culture Vessel After mounting of all parts, the top-plate has to be assembled in the reverse order.

Fig. 4 - 6: Removal /assembly of the top-plate, example for vessel type B 2

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1.

Carefully place the top-plate onto the rim of the vessel. Turn the top plate in such a way that the openings for the holding screws come to rest directly below the screws. Take care not to warp the seals.

2.

Carefully tighten the top-plate screws handtight in a crosswise manner. This will suffice to locate the top-plate and ensure the pressure for sterile connection.

Note: Do not use a spanner or other tools, since this can apply uneven pressure to the rim of the glass vessel. This may damage the vessel.

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Equipment of the culture vessels

4.3.4

Fitting of the Standard Equipment inside a Culture Vessel

4.3.4.1

Fitting, Arrangement or Rebuilding of Stirrers




The standard equipment includes 6-bladed disc impellers. Since the baffles provide additional mixing of the medium, they allow sufficient agitation for standard applications.




For special cultures, e.g. requiring low stirring speeds, special stirrers (paddle stirrers, etc.) can be fitted. These may be fermentation processes using cells, which are sensitive to shear forces, such as tissue cells, for instance. The standard paddle stirrers are available with two large blades of fixed angle of adjustment (45°). The vessel B10 and (optional) all other vessels can be equipped with stirrers with adjustable paddles. Type, for culture vessel

stirrer Ø Da

Cat. - No.

6-bladed disc impellers B2

53

3821785/6

6-bladed disc impellers B5

64

3821855/0

6-bladed disc impellers B10

70

3821488/1

Paddle stirrers B2

58

3823907/8

Paddle stirrers B5

70

3823954/0

Paddle stirrers B10

110

884 054/7

Mounting or rebuildung of the stirrers: 1.

In order to adjust the height of the stirrer elements, loosen the screws, which locate the stirrers at their position on the shaft and move the stirrers to the intended height.

2.

In order to fit other stirrers, remove the old one's and push the new onto the stirrer shaft to the required extent.




For the paddle stirrers it is recommended to locate them in crosswise arrangement (i.e. to lower stirrer turned by 90° compared to the upper stirrer).

3.

Tighten the screws in such a way that they cannot loosen even when stirring is carried out at high speeds, with highly viscous culture media or for long fermentation runs.

Fig. 4 - 7: Mounting or rebuilding of the stirrers

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Equipment of the culture vessels

4.3.5

Baffle Insert and Baffles for Culture Vessels with Stirring Device

The baffle insert of the culture vessels B2 and B5 and the two baffles of the vessel B10 serve to increase the turbulance of the culture broth, to improve the mixing and mass transfer even at low stirrer speeds. The baffle insert consists of a holder with four vertically mounted baffles. For the culture vessel B10 one baffle is attached to the aearation pipe and one is mounted into a 6 mm top-plate port (not shown in herein, see your vessel).


Specifications of baffle inserts and single baffles

Type, for culture vessel

baffle insert B2 cat.- no. 38219638

baffle insert B5 cat.- no. 38219670

sparger pipe with baffle, cat.- no. 38241080 single baffle B10, cat.- no. 38241072

Ø Da [mm]

125

154

--

HB [mm]

130

204

280

Htot [mm]

200

300

433

O-ring tightening nut

Fig. 4 - 8: Assembly of baffle insert

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7,65 x 1,78 EPDM, cat.- no. 39120945 hexagon nut M10x1, cat.- no. 38337185




On delivery the baffle insert for the culture vessels B 2 and B 5 and the baffles for the culture vessel B 10 are preinstalled in the factory. You will have to remove them only for cleaning and for checking and replacing the O-ring. You can remove them at all if you need to mount other accessories into the corresponding port.




The baffle insert of the vessels B 2 and B 5 is usually located in port no. N 15, see fig. „Top plate with recommended placement of the accessories“). For the vessel B 10 one baffle is attached to the sparger pipe, the others are mounted in separate top-plate ports.

1.

To remove the baffle insert or the baffle, unscrew the hexagon nut and pull the adapter out of the port. Check the Oring and replace, if necessary.

2.

To mount again push the extension rod with the adapter up through the topplate port, see fig. left side. Center the baffle insert in the glass vessel.

3.

Use the box spanner supplied with the tool kit to carefully tighten the adapter with the corresponding hexagon nut.

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Operating Manual BIOSTAT B

Equipment of the culture vessels

4.3.6

Sparger Pipe

The sparger pipe is made of stainless steel and has a sparger ring with evenly distributed fine holes through which the air or gas can bubble into the culture broth.


Specifications for culture vessel cat.-no. HE [mm]

B2 38217864

B5 38218569

B 10 38241080

B 2 - Airlift 38219395

B 5 - Airlift B318270200104

229

39

453

411

customized

O-ring (2)

7,65 x 1,78 EPDM, cat.-no. 39120945

tightening nut (3) threaded pin (4)

hexagon nut M10x1, cat.-no. 38337185 39504133

38318334




On delivery the sparger pipe is preinstalled in the factory. You will have to remove it only for for cleaning and checking and replacing the O-ring. At culture vessel equipped with silicone membrane insert for bubble-free gassing, the sparger pipe is not installed. See detailed information about the silicone membrane insert below.




The sparger is usually located in port no. N 14, see fig. „Top plate with recommended placement of the accessories“). If this is different for your fermentor, you can mark the placement at the top-plate or in a copy of the above figure. 1.

To remove the sparger pipe, unscrew the hexagon nut (3) and pull the adapter (1) out of the port. Check the O-ring (2) and replace, if necessary.

2.

For cleaning of the sparger ring unscrew the threaded pins (4) You can flush the pipe and ring with water. If the small holes in the ring are blocked, you can use a brush or a needle.

3.

For assembly push the sparger pipe up through its top-plate port. The adapter (1) determines the immersion depth. Center the sparger ring in relation to the stirrer shaft. If necessary, readjust the position of the lowest stirrer in such a height that the sparger ring is centered approx. 1 cm below the stirrer.




For an airlift vessel you should center the sparger ring below the guiding tube.

4.

Use the box spanner supplied with the tool kit to carefully tighten the adapter with the corresponding hexagon nut.




After comlete assembly of the culture vessel and mounting of the top-plate you can connect the silicone hose for the air supply. This can be done when you are finally equipping the culture vessel and prepare it for autoclaving.

Fig. 4 - 9: Assembly of sparger pipe

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Equipment of the culture vessels

4.3.7

Inoculation Connectors (Septum Port Kits)

Inoculation connectors (septum port kits) allow for sterile access to the culture vessel, when the seed culture or corrective solutions should be supplied. The inoculation connector B/MD is available for all culture vessels. The septum port kit B10 has been developed for the culture vessel B10 but can be installed into any top-plate port of Ø 19 mm. External supplies can be connected using a 1-channel inoculation kit. The septum membranes are „self-sealing“ and can be punctured serveral times. Part, accessories

Cat.-no.

Inoculation connector B

884 431/3

– O-ring – O-ring – septum membrane

39120954 39121054 39220672

septum port kit B10

884060/1

– septum membrane

39220486




Specifications

Notes

assembly in top-plate port Ø 6 mm 7,65 x 1,78 (EPDM) seals adapter vs. top-plate port 12,42 x 1,78 (EPDM) seals and locates cap latex self sealing after being punctured assembly in top-plate port for 1-channel inoculation kit cat.Ø 19 mm no. 884061/0 bromobutyl rubber self sealing after being punctured

Use new septum membranes for assembly of the adapters for a new process. Check all Orings of the adapters and clean and replace, if required. Assembling the septum port B/MD: 1.

Push the adapter into the top-plate port from below and screw tight with the nut.

2.

Lay septum membrane onto the adapter. Tighten carefully handtight with locating sleeve.

3.

Put sterile cap onto the locating sleeve. This cap will keep the septum membrane sterile after autoclaving until the port is required for inoculation.

Fig. 4 - 10: Fitting an inoculation connector (septum port) Assembling the septum port B10 1.

Lay the septum membrane (2) into the topplate port (1). Insert the membrane locating nut (3) and screw tight to locate the membrane.

2.

Insert the blind closure (4) into the adapter and screw tight. It keeps the membrane clean until you connect the 1-channel inoculation kit for transfer of the seed culture or the corrective solution.

Fig. 4 - 11: Septum port kit B10 with blind closure

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Equipment of the culture vessels

4.3.8

1-Channel Inoculation Kit

Similar to the inoculation kit B/MD the 1-channel inoculation kit allows for sterile access to the culture vessel, when the seed culture or corrective solutions should be supplied. A special advantage is the use of larger tubings for an increased flow of medium and the supply of larger volumes. The kit requires a top-plate port of Ø 19 mm equipped with a septum port kit B10.

Part, accessories 1-channel inoculation kit – 1-channel inoculation adapter – O-ring – silicone tubing

Cat.-no.

Specifications

Notes

884061/0 B300940100104

for septum port B10 hollow needle Ø 6 x 0,5 mm of stainless steel 1.4435 15,6 x 1,78, (EPDM) Ø 3.2 x 1.6, silicone rubber

incl. sterile sleeve

39120830 39971155

delivery set of 2 m

Preparing of the 1-channel inoculation kit

Fig. 4 - 12: 1-channel inoculation kit

BAE_B Rev. 2.31 - 1099 SW 4.1

1.

Check and clean a previously used 1channel inoculation kit. No residues of medium should glue inside the needle (3) and internal pipe. Check the O-ring and clean or replace, if necessary.

2.

The sterile sleeve is filled with glass silk (7) which can get moistened and dirty by time. This can hinder proper deareation during sterilization. Therefore you should check the filling regularly. Unscrew the screw cap (6) and check and replace the glass silk, if required.

3.

Attach the silicone tubing for medium transfer to the hose connector (4). Cut as long as required and connect to the corrective solution bottle or the container for the seed culture.

4.

Screw on the sterile sleeve. It will keep the needle (3) of the inoculation kit sterile, until it is connected to the septum port for supply of the seed culture or of the corrective solution.

5.

Prepare the corrective solution bottle or the container for the seed culture and sterilize together with the 1-channel inoculation kit and the tubing.




For connection of the 1-channel inoculation kit to the septum port see the connection information below.

4 - 16

4

Operating Manual BIOSTAT B

Equipment of the culture vessels

Connection of the 1-channel inoculation kit:

Fig. 4 - 13: Connection of the 1-channel inoculation kit

BAE_B Rev. 2.31 - 1099 SW 4.1

1.

Remove the blind closure (6) from the membrane locating nut (7). Flame the port with a flame to prevent contaminations of the membrane.




You may also wet the membrane with a disinfectant and allow to take effect for some minutes.




Note: The disinfectant must not be introduced into the vessel while you puncture the membrane with the needle. It may interfere with the process, then. It can also contain resistant spores which will then contaminate the culture.

2.

Unscrew the sterile sleeve (5) from the inoculation kit (1). Also flame the needle (3) to prevent contaminations.

3.

Puncture the membrane (6), push through the needle vertically and screw tight with the screw (2) in the membrane locating nut (7). Then transfer the seed culture (or the corrective solution, if applied) into the culture vessel.

3.

You can leeve the inoculation kit screwed into the membrane locating nut (7). Then clamp-off the tubing.

4.

If you want to remove the needle for connection of other supplies lateron : – remove needle from the membrane – flame needle and port (membrane) – flame blind closure or the needle of the new kit and screw tight in the port

4 - 17

4

Operating Manual BIOSTAT B

Equipment of the culture vessels

4.3.9

Universal-Adapter

The universal adapter allows to use single top-plate ports for connection of the supplies of corrective solution, for instance, if all other port are use for asseblies. In addition you can connect a headspace dearation, when the culture vessel is gassed and deaerated via the silicone membrane insert for bubble-free gas supply, see below for details. Part, accessory

Cat.-no.

Specifications

Notes

universal adapter (1)

3833697/9

hose connector 4 mm for tubings of Ø 1.6 x 1.6

free 6 mm top-plate port required

tightening nut (2)

38337185

hexagon nut M10x1

O-ring (3)

3912094/5

7,65 x 1,78 (EPDM)

Assembling information 1.

Check the O-ring (3) and clean or replace, as required.

2.

Insert the adapter (1) into a top-plate port Ø = 6 mm from below and screw tight with the hexagon nut (2).

3.

For corrective solution supply connect tubing (1.6 x 1.6) of the required length to the hose connector (2).

4.

If to be used for dearation of the headspace connect a sterile filter via a short piece of tubing to the hose connector.

Fig. 4 - 14: Universal-Adapter 4.3.10

Quadruple Addition Port B

The quadruple addition port allows for additional connection of up to four corrective solution supplies to the culture vessel, if the other hose connectors are occupied. Part, accessories

Cat.-no.

Specifications

Notes

quadruple addition port

884 431/3

hose connectors for tubings of Ø 1.6 x 1.6

top-plate port of Ø 19 mm required for assembly

O-ring no.

39120830

15,6 x 1,78 (EPDM)

Assembly and connection 1.

Check the O-ring (3) and replace, if required. Screw the adapter (1) into top-plate port Ø = 19 mm

2.

Connect the tubings of the corrective solution supplies to the hose connectors (2). If any of the hose connectors should not be used you can attach a short piece of silicone tubing and make a knot into the tubing.

Fig. 4 - 15: quadruple addition port

BAE_B Rev. 2.31 - 1099 SW 4.1

4 - 18

4

Operating Manual BIOSTAT B

Equipment of the culture vessels

4.3.11

Reference Thermometer

A reference thermometer allows for visual control of the temperature in the culture vessel. The thermometer will be inserted into a pocket, which is to be mounted into the top-plate while the top-plate is removed. The pocket and the housing protect the glass thermometer against damages. If a thermometer is damaged during the process, the pocket prevents the culture from being contaminated. The thermometer may be autoclaveable or not. An autoclaveable thermometer can be inserted into the pocket even before the culture vessel is sterilized. Else you will have to leave the thermometer off the pocket until you are going to start the fermentation run. Reference thermometer, cat.-no. for culture vessel type

884 4321 B2

884 4534 B5

884 4534 B 10

884 4534 B 2-Airlift

884 4534 B 5-Airlift

HH [mm]

297

297

297

297

297

HE [mm]

200

300

300

300

300

33045054

33045061

33045061

33045061

33045061

glass thermometer O-ring

2 O-rings 7,65 x 1,78 EPDM, cat.- no. 39120945

Assembling information: 1.

Push the insertion (1) pocket into the top-plate port from below.

2.

Screw tight the pocket in the port with the hexagon nut (5).

3.

Insert the thermometer (4) into the pocket.




If the thermometer is autoclaveable, you can do this prior to the sterilization. If not, insert the thermometer, when visual temperature monitoring should be applied for the process.

4.

Place the protective housing(6) over the thermometer and screw tight.

Fig. 4 - 16: Reference thermometer with insertion pocket (1) insertion pocket (2) O-rings (protective housing, adapter of insertion pocket) (4) glass thermometer (5) hexagon nut (6) protective housing

BAE_B Rev. 2.31 - 1099 SW 4.1

4 - 19

4

Operating Manual BIOSTAT B

Equipment of the culture vessels

4.3.12

Harvesting Pipe

The height-adjustable harvesting pipe is the standard equipment of the culture vessels of the BIOSTAT® B for harvesting of the product at the end of your process. It can also be used for removal of samples. Information about other devices, which can be used for sampling, is given further below. Cat. - no. for culture vessel type

38337762 B2

38342731 B5

38345331 B 10

38339633 B 2 - Airlift

38342731 B 5 - Airlift

180

310

410

380

380

HE, max. [mm] adapter (2) locking ring (7)

adapter for harvest pipe, cat.- no. 38337630 6 x 0,7 DIN 471-A2, cat.- no. 39507521

clamping cone (5)

cat.- no. 38337649

screw cap (6)

cat.- no. 38336820

O-ring (3)

7,65 x 1,78 EPDM, cat.- no. 39120945

O-ring (8)

6,07 x 1,78 EPDM, cat.-no. 39120821

nut (4)

hexagon nut M10x1, cat.- no. 38337185

Assembling the harvest pipe:

Fig. 4 - 17: Fitting the harvest pipe

1.

Check the O-rings (3) and (8). Clean or replace, if necessary. The O-ring (3) seals the adapter against the top-plate port, the O-ring (8) seals the pipe against the adapter.

2.

Insert the adapter for the harvest pipe (2) into the top-plate port (N12 recommended) and screw tight with the hexagon nut (4). Slide clamping cone (5) and screw cap (6) onto the harvest pipe.

3.

Insert the harvest pipe into the adapter and push through until its lower end is at the intended insertion depth. The maximum insertion depth is achieved if the locking ring (7) is located at the adapter.

4.

Tigthen the harvest pipe carefully handtight with the screw cap (6). The clamping ring locates the pipe at the intended insertion depth.

5.

Connect a piece of silicone tubing to the hose connector of the pipe. You should consider the required length for connection to the container for the samples or the harvested product and whether the tubing should be connected to a pump.

Caution: Never push the harvest pipe deeper into the vessel after sterilization. This may cause infections by environmental germs.

BAE_B Rev. 2.31 - 1099 SW 4.1

4 - 20

4

Operating Manual BIOSTAT B

Equipment of the culture vessels

4.3.13

Harvest Filter for Removal of Medium at Microcarrier-Cultures

The harvest filter is an optional device for removal of samples in cultures of suspended cells and with cells growing on microcarriers. It consists of harvesting pipe combined with a special harvesting filter (teflon element of pore width of 25 µm). Depending on the size of the cells the filter allows the medium and cell debris (if occuring) to permeate through the teflon element and be removed through the pipe while the the suspended cells or the microcarriers with the cells are retained in the vessel.


Specifications of harvest filter

for culture vessel type cat.- no. adapter

B2

B5

B10

884 438/0

884 457/7

884 050/4

38337630, adapter for harvest pipe

clamping cone

38337649

screw cap

38336820

O-ring

7,65 x 1,78 EPDM, cat.- no. 39120945

O-ring

6,07 x 1,78 EPDM, cat.-no. 39120821

nut

hexagon nut M10x1, cat.- no. 38337185

Assembling the harvest filter 1.

Insert the harvest pipe into the top-plate port from below, where the corresponding adapter with clamping cone is installed (port N12, for instance) . Arrange the pipe at the intended immersion depth, so that the stirrers cannot hit against the filter tube.

2.

Locate the harvest pipe with the screw cap carefully handtight.

3.

Connect a piece of silicone tubing of the required length for connection to the sampling container to the hose connector of the pipe. You should consider, whether the tubing need to be connected to a pump for sample removal.

Maintenance information


The teflon filter tube can be blocked by cell debris and macromolecules.

1.

For cleanig remove the harvest pipe from the top-plate. Connect the inlet of the pipe to a supply of water and flush by counterflow.

2.

If the layer and blocking cannot removed by this, you can unscrew the teflon tube from the pipe and clean using a brush, for instance.

BAE_B Rev. 2.31 - 1099 SW 4.1

4 - 21

4

Operating Manual BIOSTAT B

Equipment of the culture vessels

4.3.14

Spinfilter

Spinfilters are used for removal of medium while suspended cells or microcarriers are retained in the vessel. The sleeve consists of a 4-layer square mesh made of stainless steel 1.4404. There are different mesh sizes available. The mesh size determines which particles can permeate and which are retained in the culture. Spinfilters are often used in tissue cell cultures, where microcarrier-bound cells or suspended cells should be retained, while medium and suspended cell debris should be removed. Equipment for culture vessel

B2

B5

B10

spinfilter pore size 20 µm, cat.-no.

880 830/9

880 833/3

884 055/5

spinfilter pore size 40 µm, cat.-no.

880 831/7

880 834/1

884 056/3

spinfilter pore size 75 µm, cat.-no.

880 832/5

880 835/0

884 057/1

9.25 x 1.78, 39120953

on request

14.0 x 1.78, 39120767

2 x 3823907/8

2 x 3823954/0

2 x 884 054/7

880 826/0

880 827/9

884 059/8

locating pin (5) O-ring (4), size, cat.-no. paddle stirrers, number, cat. -no. harvest pipe SF (7), not adjustable

The spinfilter will be attached to the stirrer shaft. The immersion depth is to be adjusted so that the upper edge of the insert is kept above the liquid level (the medium must not flow over the upper egde). You will mount a special harvest pipe which reaches into the inner compartment of the insert. Before assemby we recommend to label the filling level for the working volume on the culture vessel. Assembling of the spinfilter:

Fig. 4 - 18: Assembly of the spinfilter: (2) stirrer shaft (3) paddle stirrers (4) O-ring (5) locating screw (6) Spinfilter (7) harvest pipe SF

BAE_B Rev. 2.31 - 1099 SW 4.1

1.

Remove the top-plate and loosen the stirrers. Pull them off the shaft.

2.

Mount the harvest pipe SF into one of the inner top-plate ports, so that it can reach into the inner compartment of the spinfilter after assembly (for this the harvest pipes for the culture vessels B 5 and B 10 have an angular pipe; the fig. shows the straight pipe as used for the culture vessel B 2).

3.

Slide the spinfilter onto the shaft and up to the intended height. Consider the filling level for this. In addition, the lower end of the harvest pipe should come close to the bottom of the spinfilter. Locate the spinfilter with the pin (5).

4.

Slide the paddle stirrers onto the shaft. You should arrange the stirrers crosswise (i.e. the upper stirrer should be turned by 90° compared to the lower stirrer. The lower stirrer should come close to the bottom of the culture vessel.

5.

After assembly of the culture vessel connect the tubing for medium removal to the harvest pipe SF.

4 - 22

4

Operating Manual BIOSTAT B

Equipment of the culture vessels

Examples for setting-up of a culture vessel with a spinfilter and the accessories:


left hand figure: spinfilter in a standard culture vessel B2, as used for cultures of microcarrier-bounded microorganisms and cells, for instance;




right hand figure: spinfilter applied together with a silicone membrane gas supply insert, as used for tissue cell cultures, for instance.

Fig. 4 - 19: Spinfilter in standard vessel B2: (1) glass vessel (2) stirrer shaft (3) paddle stirrers (in crosswise mounting) (5) locating screw (6) spinfilter (7) harvest pipe SF (8) level probe

BAE_B Rev. 2.31 - 1099 SW 4.1

Fig. 4 - 20: Assembly of a spinfilter together with a silicone membrane insert: (2) stirrer shaft (3) paddle stirrers (crosswise mounting) (5) silicone membrane insert (6) windings of silicone membrane (7) harvest pipe Sffor vessel B2 (8) level probe (9) holder for silicone membrane (10) connection of gas inlet (11) connection of exhaust (13) spinfilter

4 - 23

4

Operating Manual BIOSTAT B

Equipment of the culture vessels

4.3.15

Silicone Membrane Insert

A silicone membrane insert is an assembly providing bubble free gas supply to the culture vessel. It is often used in tissue cell culture or cultures with cells, which are sensitive to shear forces. The membrane insert is installed instead of the sparger pipe. The entire kit includes the following parts: silicone membrane insert, cat. -no., for culture vessel type silicone membrane, delivered length cat. -no. paddle stirrers, cat. -no.

884 466/6 B2

884 467/4 B5

884 048/2 B10

3 x 3.7 / 5,2 m 3997144/9

3 x 3.7 / 10,4 m 3997144/9

3 x 3.7 / 16 m 3997144/9

2 pcs., 3823907/8

3 pcs., 3823954/0

3 pcs., 884 054/7

membrane filter gas in-/outlet delivery set, cat. -no.

PTFE membrane Ø 50 mm, pore width 0,2 µm, filter area 2 19.6 cm , 2 x 881 007/9

pressure controller

setup acc. to drwg. B231.20.08.00.104, order on request

The membrane is optimized for reversible gas diffusion of O2, N2 und CO2. For gas supply you can use technical gasses or a Gas Mix Unit, cat.-no. 884 171/3, where you can adjust a mixture of gasses optimum for the process. O2 and N2 allow a reversible pO2-control. CO2 can be supplied for pH-control while excess gaseous CO2 of the medium can permeate the membrane and be exhausted via the membrane outlet. The direction of gas diffusion is determined by the differential partial pressure of the gasses in the membrane compartment versus the partial pressure of the gas dissolved in the medium. The pressure inside the membrane is controlled via a pressure controller in the outlet. Assembly of the silicone membrane kit:

Fig. 4 - 21: Silicone membrane insert kit: (2) stirrer shaft (3) paddle stirrers (5) holder for silicone membrane (6) silicone membrane (7) harvest pipe (9) rod for holder assembly

BAE_B Rev. 2.31 - 1099 SW 4.1

1.

Check whether the silicone membrane is properly wrapped around the holder. It is wrapped too tight, if the membrane is depressed at the rods of the holder. It is wrapped around too loose, if it sloughes off the indents of the rods.

2.

If a membrane is damaged and need to be replaced, loosen the membrane and wrap the new membrane around the rods of the holder. It should be evenly wrapped around. The inlet side is at the lower hose connector of the holder, the outlet is at the upper hose connector. You should use hose clips to locate the membranes at the hose connectors.

3.

Mount the spinfilter, if it should be applied. See the section above and the figure on the next page.

4.

Mount the paddles stirrers to the stirrer shaft and adjust.

5.

Mount the holder with ist rod into the top-plate and locate with the hexagon nut. Recommende top-plate port is N17.

4 - 24

4

Operating Manual BIOSTAT B

Equipment of the culture vessels

6.

Mount universal hose adapters for connection of the membranes and of the gas supply and exhaust into the top-plate. Attach the inlet and outlet tubing of the membrane insert to the lower hose connectors of these adapters.

7.

Connect the membrane filter of the inlet gas to the „inlet“ hose adapter via a short piece of silicone membrane. Also connect the exhaust filter to the „outlet“ hose adapter.




For optimum gas transfer the internal pressure of the silicone membrane must be precisely adjusted. For this attach the pressure controller (12) to the vessel and connect to the exhaust filter. Gas is supplied to the silicone membrane at a given max. pressure and preadjusted flow rate. The internal pressure is then adjusted with the exhaust pressure controller.




Note: The internal pressure of the membrane must not exceed 0.8 bar (11.6 psig). Overpressure can cause the membrane to burst!

8.

After assembly, when filling the culture vessel and during operation, you should ensure that the membrane is allways fully covered with medium. If a spinfilter is installed, the medium must not flush over the upper edge of this device into the inner compartment.Therefore, after taking samples, you should refill with medium or use a level control for this.

Fig. 4 - 22:: external connections of the insert: (1) glass vessel (2) ... (9) see fig. on previous page (10) gas inlet filter (11) exhaust filter (membrane outlet) (12) pressure controller

BAE_B Rev. 2.31 - 1099 SW 4.1

Fig. 4 - 23: complete setup of a culture vessel B2, including the spinfilter in the culture vessel (13)

4 - 25

4

Operating Manual BIOSTAT B

Equipment of the culture vessels

4.3.16

Blind Closures

Blind closures need to be fitted to any top-plate port, where no other accessory, adapter or culture vessel equipment is installed.


You should note that the blind closures for top-plate ports of Ø 6 mm must be installed before the top-plate is mounted to the vessel. Those ports cannot be used for assembly of other culture vessel assemblies lateron. Therefore you must carefully check which assemblies are required for the process and need to be mounted into which top-plate port.

Product Information Blind closures, type

DN 11

PG 13,5

DN 19

cat.-no.

38337193

38344416

38316773

for top-plate port

Ø = 6 mm

Ø = 12 mm

Ø = 19 mm

7 7 7 4 8

2 2 2 1 2

2 2 4 5 3

locating screw, type, cat.-no.

M10 x 1, 38337185

38344416

38316773

O-ring, type / dimensions cat.-no.

7,65 x 1,78 EPDM, 39120945

--

15,6 x 1,78 EPDM, 39120830

--

38606127

--

number, for culture vessel: – B2 – B5 – B 10 – B 2 - Airlift – B 5 - Airlift

sealing, cat.- no.

Assembling of blind closures 1.

Fit the blind closures to those ports which will not be used for other assemblies.

2.

Insert a sealing membrane into the ports of Ø = 12 mm, before fitting the corresponding blind closure.

Note: It may be advisable to prepare ports, which are not used by assemblies, as inoculation ports or with universal adapters in order to provide sterile access to the interior of the culture vessel, when required lateron during the fermentation run.

Fig. 4 - 24: Blind closures for top-plate ports of Ø 6 mm, Ø 12 mm and Ø 19 mm

BAE_B Rev. 2.31 - 1099 SW 4.1

4 - 26

4

Operating Manual BIOSTAT B

Equipment of the culture vessels

4.4

Probes and Electrodes

4.4.1

General Notes

1.

Check the requirements for measurement of parameters of your process. Mount all probes and electrodes as shown below. For recommended placement see the fig. on page 4-9.




The immersion depth is determined by the dimensions of the probes and electrodes and their adapters. The immersion depth of the level and antifoam probe can be reduced to adapt it to the working volume of the culture vessel.

2.

Before assembling of the probes and electrodes you should check the O-rings and replace, if necessary. Only O-rings being clean and free of damages will ensure a sterile assembly. It is recommended to grease the O-rings with some silicone grease. This will prevent the Orings from glueing to their contact surfaces of the ports, which can damage them.

3.

Carefully tighten the locating screws with their hexagon nuts or screw caps with the spanners enclude in the tools kit. Knurled screwes should only be tightened carefully handtight.

4.4.2


Pt-100 - Sensor for Temperature Measurement The Pt-100 - temperature sensor will usually be mounted into top-plate port N2, see fig. on page 4-9. If this is not true with your culture vessel you should mark the port at your vessel correspondingly. The following sensors and attachments are included for the vessels: for culture vessel type cat.- no. type HE [mm]

B2, 33190887

B5 33190895

B10 00001769

B2-Airlift, 33190895

B5-Airlift, on request

200-4

300-4

400-4

300-4

300-4

211

316

418

316

316

O-ring (2) locating nut (3)

Fig. 4 - 25: Assembly of Pt-100

BAE_B Rev. 2.31 - 1099 SW 4.1

7,65 x 1,78 EPDM, cat.- no. 39120945 hexagon nut M10x1, cat.- no. 38337185

1.

For removal of an installed Pt-100, unscrew the locating nut (3) and pull the Pt-100 out of its port.

2.

If necessary clean the sensor shaft mechanically. The probe can be fouled by cells, cell debris and floated compenents of the culture broth, e.g. proteins. Replace the Oring (2), if required.

3.

Insert the Pt100 into top-plate port of the culture vessel from top. The immersion depth is determined by the adapter (1). Slide the hexagon nut (3) onto the sensor shaft from below and screw tight.




Before the sterilization of the culture vessel you should wrap some aluminium foil around the plug of the cable. This will protect the plug against the impact of steam. After the sterilization connect the Pt-100 with the multi-conductor plug to the socket on the control unit.

4 - 27

4

Operating Manual BIOSTAT B

Equipment of the culture vessels

4.4.3

Antifoam and Level Probes

The antifoam probe and the level probe are single conductive electrodes. Their measuring principle is conductivity measurement; the stainless steel parts of the culture vessel act as the opposing electrode. Foam or culture medium in contact with the probes causes a current jump by changing the conductivity of the measuring system. The probes have a long insulating ceramic coating. This coating prevents short circuit due to fouling. The following sensors and attachments are included for the vessels: probe, for culture vessel type antifoam probe, cat.- no. probe type HE [mm] level probe, cat.- no. HE [mm] O-ring, adapter vs. top-plate port O-ring, probe shaft vs. adapter

B2

B5

B 10

B 2 - Airlift

B 5 - Airlift

884446/1

884446/1

884446/1

884446/1

884446/1

MD 85

MD 85

MD 85

MD 85

MD 85

50

50

50

50

50

884448/8

884448/8

884448/8 opt. 884469/0

884448/8

884448/8

122

122

122 opt. 260

122

122

7,65 x 1,78 EPDM, cat.- no. 39120945 6,07 x 1,78 EPDM, cat.-no. 39120821 (not shown)

Maintenance : 1.

If necessary clean the probe´s shaft mechanically. The ceramic coating of the probe can partially be fouled by cells, cell debris and floated compenents of the culture broth, such as proteins.

2.

Check the O-rings of the adapter and inside the adapter (not shown in the fig. below). Clean or replace if required.

BAE_B Rev. 2.31 - 1099 SW 4.1

4 - 28

4

Operating Manual BIOSTAT B

Equipment of the culture vessels

Assembling the antifoam and level probe:

Fig. 4 - 26: Assembling the antifoam and level probe

1.

Insert the adapters for the antifoam and the level probe into their corresponding top-plate ports from below (port no. N3 recommended for the antifoam probe, N11 for the level probe) while the topplate is removed. Tighten adapter with hexagon nut using a spanner.

2.

After mounting of the top-plate onto the glass vessel insert the probes into their adapters from top down to the required height and screw tight with their locating screws.




The possible immersion depths depend on the probes delivered for the culture vessel, see above table.

3.

For adjustment of the immersion depth of the level probe you will need to measure the filling level obtained with the intended working volume. You should lable the level on the vessel correspondingly.




The O-ring inside the adapter seals the probe shaft against the adapter. Therefore it is possible to pull out the electrode a little even after sterilization for readjustment, if the immersion depth is adjusted too deep.

Caution: Never push the probe deeper into the vessel after sterilization. This may cause infections by environmental germs.

Other measures 1.

Remove the cable before placing the culture vessel with the antifoam and level probe in the autoclave. The cable must not be sterilized.

2.

After autoclaving / before starting fermentor operation connect the probes to their corresponding sockets „Foam“ and „Level“ on the left side of the control unit.




For detailed information about the adjustment of the operation parameters of the antifoam and level measurement and control see the corresponding chapters in section 5.

BAE_B Rev. 2.31 - 1099 SW 4.1

4 - 29

4

Operating Manual BIOSTAT B

Equipment of the culture vessels

4.4.4

pH - Electrode

The pH - electrodes are sterilizable combined pH-electrode assemblies, manufactured by INGOLD. For the BIOSTAT® B electrodes with pasteous bridging electrolyte are used. Such pH - electrodes are an alternative to electrodes filled with liquid electrolyte, offering several advantages :


pH-electrodes with pasty electrolyte have a pressurized electrolyte compartment and can be used in environments having an operating pressure of up to 2.5 bar (about 36 psi). The max. pressure permissible for a culture vessel of the BIOSTAT® B is 1 bar (14.5 psig)




Although a small amount of the electrolyte continuously penetrates the diaphragm, the filling suffices for the whole lifetime. The electrodes are maintenance-free.

The component parts of the combined glass electrode set up a galvanic chain where the total potential Eges (in „V“) which is the total sum of all boundary surface potentials Ei, can be measured with a high impedance voltmeter connected between discharge electrode and reference electrode. The pH value 4-1) is calculuted from measured potential Eges according to the NERNST equation . pH-electrode, cat.- no.

33187711

33187703

33187703 or 08844640

08844607

33187703

B2

B5

B 10

B 2 - Airlift

B 5 - Airlift

Pa 12/200

PA/12/325

PA/12/325 or PA/12/425

Pa 12/200

PA/12/325

HSo [mm]

200

325

325 or 425

200

325

O-ring

10.77 x 2.62 (silicone), included in the delivery of the electrode

for culture vessel type type

4.4.4.1

Reactivation of pH - Electrodes 1.

For the first use and after long-term storage of the electrode in dry state it need to be reactivated and conditioned carefully.




Glass membranes, as used for pH electrodes, built up a thin watery gel layer, due to a reaction with water. This layer essentially effects the measuring quality of the probe (response time, slope, alkali mistake) and therefore should regenerate after a long-term storage.

2.

For reactivation you should place the pH-electrode into a beaker with demineralized water for about 12 ... 14 h. See also the information in the documents delivered with the electrode.

Fig. 4 - 27: pH - electrode

4-1)

See details about the NERNST equation in the corresponding literature

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Equipment of the culture vessels

4.4.4.2

Calibration of the pH - electrode




The pH - electrode is calibrated before assembly into the culture vessel. The calibration is accomplished in two steps, „zero“ and „slope“ calibration. For details about how to proceed for calibration of the pH-electrode see the corresponding chapter in section 5.




Caution when handing the calibration buffers - they can cause burns.




To consider the possible effects of the heat impact during sterilization and of chemical and physical reactions of the electrolyte with ingredients of the culture medium, the ph-electrode can be recalibrated during the process. See also the corresponding chapter in section 5.

4.4.4.3

Assembly at the Culture Vessel

1.

Insert the pH-electrode into its top-plate port (port N13 recommended, see fig. „Top plate of a BIOSTAT® B culture vessel with recommended placement of the accessories“). The immersion depth below the top plate depends on the delivered electrode. The culture vessel should be filled with water or the culture medium time to prevent drying of the electrode.

2.

Tighten the electrode carefully handtight with. Do not use a spanner or similar tools.

3.

For autoclaving disconnect the cable. You can cover the plug of the electrode with the protective cap included in the delivery or wrap some aluminium foil around to protect against the impact of steam.

4.

After autoclaving / before starting fermentor operation connect pH -electrode probe with its cable to the „pH“ - socket at the left side of the control unit.

4.4.4.4

Maintenance

A pH - electrode is subject to wear and tear. This is caused by the thermal effects during the sterilization or by chemical reactions of the diaphragm or of the pasteous electrolyte with ingredients of the culture medium, for instance. Also fouling by cells, cell debris or macromolecules can change the measurement behavior.


The pH - electrode can be sterilized several times. The number of sterilizations depends on the process conditions (i.e. the composition of the medium and resulting impacts on the electrode). If a pH - calibration is malfunctioning, it should be replaced.




Symptoms for wear and tear are a slower response, a reduced slope or a zero drift. If the measured pH-value is different for the medium at intensive stirring and at stopped stirrer drive, this may incicate fouling of the diaphragma.




A functional check of the pH - electrode is limitted to checking the electrode´s zero and slope and to comparing the pH-value measured „online“ with the pH-value measured in a fresh sample. For details see the section „pH - Calibration“ in section 5 of this manual and the documentation delivered with the electrode.

4.4.4.5

Storage of the pH - Electrode

1.

You should flush the pH - electrode with aqua dest. after any run. Never store an electrode with dirty diaphragm. The residues will dry and may not be removable.

2.

Short-term storage is possible in demineralized water. For a longer storage you can place the pH - electrode in a beaker filled with 3m-KCL solution. You can also fill a small amount of this electrolyte into the protective cap delivered with the electrode and attach this cap onto the electrode´s tip.

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Operating Manual BIOSTAT B

Equipment of the culture vessels

4.4.5

pO2 - Electrode

The measurement of the dissolved oxygen is made with a sterilizable pO2-electrode (manufactured by INGOLD). The electrode operates on the polargraphic principle and consists of an Ag-anode and Ptcathode, which are seperated by a gas permeable polymer membrane from the solution to be measured (CLARK-principle). Anode and cathode are connected conductively by an electrolyte; the electrolyte builds a small, definable layer between the membrane and the cathode. At a suitable polarization voltage, the oxygen, which diffuses through the membrane into the cathode, will be reduced. This chemical reaction produces an electrical current in the nA-range, which is directly proportional to the oxygen partial pressure (the actual measureable quantity of the measured solution). Since the permability of the membrane depends on temperature, an exponential rinse (3 %/ degC) in the electrode current will be observed at rising temperature. This effect will be compensated by automatic temperature compensation by means of an integrated thermistor in the electrode. pO2 - electrode, cat.- no. for culture vessel type

3318241/8 3318240/0

3318240/0 opt. 884468/2

3318241/8 3318240/0

B2

B5

B 10

B 2 - Airlift

B 5 - Airlift

12/220

12/320

12/320; optional 12/420

12/220

12/320

HSo [mm]

212

312

312, opt. 412

212

312

O-ring

10.77 x 2.62 (silicone), included in the delivery of the electrode

type of electrode

Assembly of the pO2 - Electrode:

Fig. 4 - 28: Assembling the pO2 - electrode

BAE_B Rev. 2.31 - 1099 SW 4.1

1.

Insert the pO2 - electrode into the suitable port (port N4 recommended, see fig. on page 4-9). The immersion length below the top plate depends on the electrode delivered with the type of culture vessel.

2.

Screw the locating nut carefully handtight, do not use a spanner or similar tool.

3.

For autoclaving disconnect the cable. You can cover the plug of the electrode with the protective cap included in the delivery or wrap some aluminium foil around to protect against the impact of steam.

4.

After autoclaving / before starting fermentor operation connect the pO2 electrode with its cable to the socket „pO2“ at the left side of the control unit.




The electrode must be polarized for at least 6 h before calibration or measurement. See detailed information below.

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Operating Manual BIOSTAT B

Equipment of the culture vessels

4.4.5.1

Calibration of the pO2 - Electrode




Below you will find a summary of important steps of calibration. More detailed information and the inputs required for the calibration menu are included in section 5.




The calibration of the pO2 - electrode is made in the culture vessel after autoclaving and under the conditions of the fermentation. Before calibration, it must be polarized. You can use a polarization module available from the manufacturer for this or you can connect the electrode to the control unit and switch-on the system. The polarization must be repeated any time the electrode is disconnected from the amplifier for more than 10 min, but may require less time then.




Calibration includes zero and slope calibration. The „zero“ is the electrode´s current, when no oxygen is present in the culture medium, the „slope“ is usually the pO2 after saturation of the medium with air at the maximum air supply intended for the process.

1.

Adjust the operating temperature in the culture vessel.

2.

For „zero“ calibration you can measure the pO2 of the culture medium before starting the air supply. The medium will be degassed almost completely due to the heat impact during sterilization and thus should not contain dissolved oxygen. Alternatively you can s upply an oxygen-free gas (such as nitrogen of 99.98 % purity) to the culture medium to displace the dissolved oxygen until a constant pO2 near „0 %“ can be read at the measurement and control system. Confirm this value as „zero“ as shown in section 5 of this manual.

3.

For slope adjustment activate the air supply and adjust the stirring speed. The medium should be optimally gassed (max. flow rate intended for your process) and mixed. At a stable display of the measured value you can calibrate this as „100 % pO2“

4.

After calibration the gas supply rate required for the startup of the intended fermentation process can be adjusted on the rotameter of the control unit. Note that the rotameter is calibrated according to standard conditions (temperature 20 degC, with air at 2 barabs). If it is important to maintain precise operating air flow-rates for further calculations, this makes it necessary to recalculate the indicated flowrate with a correction factor. See further information the supplement.

4.4.5.2

Function Control of pO2 - Electrode

The electrode should be inspected in regular intervals. This is best carried out outside of fermentor. The electrode must be polarized at least 6 hours before the inspection. A relative good assessment can be made of the operating effectiveness of the pO2-electrode by checking the zero current and the response time, see the short information below. More detailed information will be included in the documentation of the manufacturer delivered with the electrode. 1.

Hold the electrode in the air. Then measure the pO2 and wait for a stable indication of the measured value. Note this value.

2.

Place the electrode in an oxygen-free environment (nitrogen gas of purity >99.98 %, for instance). Wait for a stable indication of the measured pO2.




When changing from ambient air to oxygen-free environment, the measured zero current must be less than 1.5 % of the current in the air after 5 minutes.




If a malfunctioning is suspected you should service the electrode. Usually the membrane module and the electrolyt will be changed for this. For detailed information on how to proceed, see the documentation of the manufacturer.




Caution when handing the electrolyte - it can cause burns.

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Equipment of the culture vessels

4.5

External Equipment for Connection to the Culture Vessels

4.5.1

Exhaust Cooler

The exhaust cooler can, by condensation, lower the water vapour content in the exhaust. This reduces the loss of liquid via the exhaust and prevents the sterile filter from being blocked by humidity.


If the exhaust filter is blocked, the air supply to the culture can be reduced or blocked at all. This can result in inadmissible overpressure.




Wet exhaust filters can be fouled by environment germs introduced via the outlet. The germs can permeate the filter membrane by time and contaminate the culture.

exhaust cooler, cat.-no.

3824042/4

3824116/1

3824116/1

3824042/4

3824116/1

B2

B5

B 10

B 2 - Airlift

B 5 - Airlift

stainless steel tube with angular pipe

stainless steel tube with straight pipe

stainless steel tube with straight pipe

stainless steel tube with angular pipe

stainless steel tube with straight pipe

222

255

255

222

255

for culture vessel type design features

HK [mm] O-Ring (1) O-Ring (2)

15,6 x 1,78 (EPDM), 3912083/0 18,77 x 1,78 39121763

29,82 x 2,62 39121780

29,82 x 2,62 39121780

18,77 x 1,78 39121763

29,82 x 2,62 39121780

nein

884 459/3

884 459/3

884 459/3

884 459/3

extension kit, cat.-no.


The exhaust cooler can be mounted directly into ist top-plate port or via the extension kit. The extension kit includes a tubing and a holder attached to the culture vessel. For the sterilization in the autoclave the exhaust assembly can be removed from this holder and bent over. This reduces the internal height required for the autoclave.

Assembly without extension kit (standard equipment, see fig. on next page) : 1.

Fit the exhaust cooler into its port in the top plate (port no. N1 recommended) and fasten it there with its cap nut.

2.

Attach the exhaust filter to the exhaust cooler via a short piece of silicone tubing as shown in the corresponding section below. Do not clamp-off this tubing for the sterilization in the autoclave. The exhaust outlet provides the necessary pressure compensation of the interior culture vessel with the ambient air.




The hoses for the cooling water supply and drain can be connected when fermentation process is initiated.

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Operating Manual BIOSTAT B

Equipment of the culture vessels

Assembly with extension kit (optional equipment) :

Fig. 4 - 29: Fitting the exhaust cooler (version for vessel type B2 with angular connection pipe)

1.

Place the tubing adapter of the extension kit into the top-plate port assigned for the exhaust cooler (port N1 recommended) and screw tight.

2.

Replace the knurled top-plate screw next to the port for the exhaust cooler by the special screw of the extension kit. This screw has a locating pin for the support rod of the extension kit, where the tubing adapter will be located.

3.

Place the support rod onto the pin and screw tight with the lower fastening screw.

4.

Place the rod attached to the upper screw adapter of the extension tubing into the boring of the support rod and screw tight with the upper fastening screw.

5.

Screw the exhaust cooler into the upper screw adapter of the extension tubing.

6.

Attach the exhaust filter to the exhaust cooler via a short piece of silicone tubing as shown in the corresponding section below. Do not clamp-off this tubing for the sterilization in the autoclave. The exhaust outlet provides the necessary pressure compensation of the interior culture vessel with the ambient air.




The hoses for the cooling water supply and drain can be connected when fermentation process is initiated.

Bending-over of the exhaust cooler for autoclaving: 1.

Loosen the screw of the support rod, which locates the rod attached to the upper screw adapter of the extension tubing. Pull the rod out of the boring of the support rod.

2.

Bent-over the exhaust cooler with the filter to minimize the height of the entire culture vessel assembly.

3.

After autoclaving place again the rod attached to the upper screw adapter of the extension tubing into the boring of the support rod and locate with the screw.

Fig. 4 - 30: Assembling of the extension kit

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Equipment of the culture vessels

4.5.2

Sterile Filters for Air/Gas Supply and Exhaust

The air supply and exhaust filters are autoclaveable membrane filters in plastic housing. They have a pore size of 0,2 µm and ensure sterile air (or gas) supply to the culture vessel and sterile exhaust. for culture vessel membrane filter delivered no., cat. -no.

B2

B5

B10

B2-Airlift

B2-Airlift

2 pcs. 881 007/9

2 pcs. 881 007/9

air inlet: 1 x 881 007/9; exhaust 1 x filter element cat.-no. 3922830/4 (not shown)

2 pcs. 881 007/9

2 pcs. 881 007/9




Filter 881 007/9




Filter 3922830/4 :

4.5.2.1

:

-

flat membrane made of PTFE, in plastic housing pore width 0,2 µm membrane - Ø = 50 mm 2 filter area 19,6 cm

- stareshape folded PTFE - membran cylinder in plastic cartridge - pore width 0,2 µm 2 - filter area about 500 cm

General Notes

1.

The membrane filters can be sterilized several times and therefore be used for several fermentation runs. The number of uses will depend on the application conditions.

2.

You should care, that the filters are not applied to vapour or moisture. If moisture condensates at the filter membrane, this may block the filter. This may require to carefully dry the filter before it can be used again. You should also avoid that foam of the culture enters the filter. Then the filter will be blocked permanently and must be replaced.

3.

Carefully check the pieces of silicone tubing used for connecting the filters. They must not be damaged, since damages can cause environmental germs to enter the tubing and contaminate the culture.

4.

Air or inlet gas can be connected and supplied from the control unit or an extra Gas Mix Unit. An extra Gas Mix Unit can be applied, if the inlet air should be enriched with oxygen.




Above this you can apply a pO2 - control by reversible supply of either O2 and N2. pH-control is possible by supply of gaseous CO2, for instance. Such control strategies are often applied for tissue cell cultures using the silicone membrane insert described above in this manual. For connection of a Gas Mix Unit see the documentation delivered with the device.

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Operating Manual BIOSTAT B

Equipment of the culture vessels

4.5.2.2

Connecting the Inlet Air Filter 1.

Connect a short piece of silicone tubing (4x7 mm) to the hose connector (2) and to the sparger pipe (or the inlet of the silicone membrane insert, if installed).

2.

Attach a piece of silicone tubing of suitable length for connection to the control unit or to the Gas Mix System onto the hose connector (1) of the filter. Secure the tubing against slipping.

3.

Connect a piece of silicone tubing of suitable length for later connection to the external supply at the inlet connector of the filter (1).

4.

Clamp-off the tubing prior to autoclaving of the culture vessel. The tubing can be inserted into the clamping device assigned for this (the „dovetail clamp“, see fig. „Top plate design and placement of accessories“ for details).

1

2

Fig. 4 - 31: Membrane filter for air inlet and exhaust, cat.-no. 881 007/9 4.5.2.3

Attaching the Exhaust Filter

1.

Connect a short piece of silicone tubing (3.2 x 1.6 mm) to the hose connector (2) of the filter and to the exhaust cooler. Secure the tubing against slipping. For a standard setup you need not install a tubing to the „outlet“ connector (1) of the exhaust filter. This provides free exhaust into ambient air.

2.

If a silicone membrane insert is used for bubble free gas supply to the medium, fit the tubing to the filter (hose connector (2) in this case) and to the outlet hose connector of the silicone membrane insert. Connect the outlet of the exhaust filter (hose connector (1)) to the pressure controller. See description of the setup of a silicone membrane insert for details.

3.

If you want to dearate from the vessel headspace, you can connect an additional exhaust filter to a hose connector of an universal adapter installed in the top-plate, for instance.




The exhaust tubing must not be clamped-off during the sterilization, since it provides the necessary pressure compensation between the culture vessel and the ambient air during and after the sterilization. The exhaust filter guarantees sterile pressure compensation.

4.

Take special care that the tubings are not damaged and do not slip during manipulations of the culture vessel. If unsterile air enters the air inlet or exhaust tubing, the culture will be contaminated by environmental germs.

5.

Also check the exhaust filter connection and the exhaust filter after the sterilization. If the culture medium tends to foam, the foam can enter and block the exhaust. Then it may be necessary to replace the filter and repeat the sterilization.

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Operating Manual BIOSTAT B

Equipment of the culture vessels

4.5.3

Corrective Solution Bottles

For corrective solutions (acid, alkaline, antifoam or nutrient solution) the delivery set of the culture vessels includes 250 ml bottles, cat.-no. 0882360/0, for the vessels B 2 and B2-airlift and 500 ml bottles, cat.-no. 0882361/8, for the vessels B 5, B5-airlift and B10. The bottles are autoclaveable glass bottles made of borosilicate glass. For supply of solutions during long-term or continuous operation, we recommend to prepare several bottles to have sufficient volumes available. If „bulk“ volumes of nutrient solutions should be supplied, storage bottles made of polypropylen may be applied (not shown herein). They offer storage volumes of 10 l (cat.-no. 882364/2), 20 l (cat.-no. 882365/0) or 50 l (cat.-no. 882366/9). Equipment of the bottles


Stainless steel cap (3) with 2 integrated hose connectors. A gasket (2) on the rim of the glass bottle (1) seals the cap. The cap is tightened with the screw cap (4).




PTFE dispenser hose (7) which is inserted into the bottle and is resistant to the aggressive action of acids and bases at high temperatures.




Sterile air filter (5), cat.-no. 3922480/5, for pressure compensation when solution is removed (1).




Silicone tubing (6) for connecting the bottle to the culture vessel.

Assembly of a bottle :

Fig. 4 - 32: Storage bottles of 250 ml for acid, alkali and antifoam solution

BAE_B Rev. 2.31 - 1099 SW 4.1

1.

Attach the PTFE hose (7) to one of the hose connectors from below. Shorten it so that the end is 1-2 mm above the bottom of the bottle.

2.

Fill the bottle with acid, alkali or antifoam agent. Screw the cap (4) onto the bottle.




Warning: When using acid or alkali agents take precautions against burns.




We recommend not to use hydrochloric acid (HCL) for pH-control. HCL can cause corrosion even of stainless steel parts.

3.

Cut a length of silicone tubing sufficiently long to connect the bottle to the control unit (for passing through the peristaltic pump). Attach it to the hose connector carrying the PTFE hose.

4.

Connect the other end of the silicone tubing to one of the nozzles for corrective solutions. Locate the tubings on the hose connectors.

5.

Attach the air filter (5) to the other hose connector via a piece of silicone tubing.

6.

Place the bottle into the holders at the culture vessel. Clamp-off the tubings. No solution should escape from the bottles during sterilization.

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Operating Manual BIOSTAT B

Equipment of the culture vessels

4.5.4

By-pass Sampler Kit

The by-pass sampler can be used for the continuous or discontinous removal of very small samples from the culture for the purpose of off-line-analysis, where almost actual samples are required. It can also be used for introducing small volumes of addition solutions which should be fed to the culture. The by-pass sampler consists of a two-channel screw connector which is fitted into the top plate of the culture vessel, the membrane holder and approx. 2 m of 1.6 x 1.6 mm silicone tubing for the by-pass hose. The membrane holder includes a septum membrane, which can be punctured using a syringe (since the membrane is self-sealing, it can be punctured several times, if necessary). You can use dosing pump such as the FE 411 (B. Braun Biotech International GmbH) to remove the sample from the culture vessel. The sample will be fed through the bypass sampler and returned to the culture vessel after it has passed through the by-pass via the second hose connector of the twochannel screw connector. Thus you will receive representative samples for the state of process, for instance.

Assembly and connection 1.

Add the rising pipe (piece of teflon tube) to one of the lower hose connectors of the adapter. During equipment of the culture vessel screw the adapter into a suitable port in the top-plate.

5.

Connect two pieces of silicone tubing to the upper hose connector: - one for sample removal from the vessel is to be connected to the hose connector with the rising pipe. - one ifor return of sample which has passed through the bypass, is to be connected to the free hose connector.

2.

Prepare the membrane holder. Insert a new piercing membrane. Add the washer and locate with the sleeve nut. Finally add the protective cap for sterilization.

3.

Connect the tubings for sample remomal and return to the membrane holder.

4.

Locate all parts for safe handling during autoclaving. Autoclave together with the culture vessel.

Fig. 4 - 33: Two-channel adapter for connection to the top-plate

Fig. 4 - 34: Assembly of membrane holder

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Operating Manual BIOSTAT B

Equipment of the culture vessels

Taking Samples using the Bypass - Sampler

Fig. 4 - 35: Taking a sample via the membrane holder

1.

After placement of the entire assembly at the working place and/or for taking samples, introduce the removal tubing into a peristaltic pump. Switch-on the pump to have a continous flow of culture broth through the membrane holder.

2.

Prepare a sterile syringe for the sample.

3.

Remove the cap of the membrane holder. Burn-off the membrane and the needle of the syringe with an open flame or moisten with a desinfectant.

4.

Pierce-in the syringe into the membrane and pull the plunger to remove the sample. Afterwards withdraw the syringe from the membrane and attach again the cap to keep the membrane sterile until you pierce-in another syringe.

Notes:


After applying a desinfectant, wait for a sufficient time so that it can take effect. Carefully check if it could be used with your culture. Residues of the desinfectant entering the medium must not influence the culture. Alcohol, for instance, may not destroy spores of some environmental germs and can cause that such spores to enter and contaminate the medium. Remove any residues of the desinfectant.




Introducing an additive solution is done in a similar way. Instead of removing the sample from the bypass, you will have to introduce the addittive solution with the syringe. Prepare a sterilie additive solution for this.




The membrane of the Bypass-Sampler is „self-sealing“ and can be punctured several times. If you are manipulating the sterile parts carefully, there is only a minimum risk of contamination of your culture.

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Operating Manual BIOSTAT B

Equipment of the culture vessels

4.5.5

Manual Sampling Kit

The manual sampler, cat.-no 884 462/3, can be used for discontinuous removal of samples at a larger volume. It consists of the following parts, see fig. of the setup:


Sampling tube (pos. 2),




Clamping device for assembly to one of the stands of the culture vessel




Syringe for sample removal with sterile filter assembly (pos. 4)




Silicone tubing 1.6 x 1.6 mm for connection of the syringe and 3.2 x 1.6 mm for connection to the harvesting pipe in the culture vessel and the sample outlet,




Hose clamp

The sampling tube has a screw cap with three hose connectors. One is used for connection to the sampling tube of the culture vessel (pos. 1), one for outlet of the sample to external receptacle (pos. 5) and one for the syringe (pos. 3). The sterile filter assembly of the syringe prevents insterile air to enter and contaminate the device. Assembling the manual sampling kit

Fig. 4 - 36: Manual Sampler

BAE_B Rev. 2.31 - 1099 SW 4.1

1.

Unscrew the cap of the sampling tube. Attach a short piece of silicone tubing Ø 3.2 x 1.6to the rising pipe, it should be reach down to the bottom of the tube. Screw cap onto the tube again.

2.

Attach a piece of silicone tubing Ø 3.2 x 1.6 to the outside hose connector of the rising pipe. This tubing is used to transfer a sample into an external receptacle (via outlet tubing, 5).




Attach another piece of silicone tubing Ø 3.2 x 1.6 to hose connector (1) and connect to the sampling pipe of the culture vessel. This tubing is used for transfer of the sample from the harvest pipe into the sampling tube

3.

Mount sterile filter to the syringe and connect to the third hose connector of the sampling tube via a short piece of silicone tubing Ø 1.6 x 1.6, see pos. (3).

4.

Attach the clamping device to one of the stand rods of the culture vessel so that the manual sampler can easily be handled. Insert the sampling tube and the syringe into the clamping device.

5.

Clamp-off the silicone tubing between the rising pipe of the culture vessel and the sampling tube prior to autoclaving.

6.

Autoclave the complete assembly together with the culture vessel.

4 - 41

Operating Manual BIOSTAT B

4 Equipment of the culture vessels

Removal of Samples with the Manual Sampler 1.

Prior to taking a sample push down the plunger of the syringe. The outlet tubing (5) should be open at this time.

2.

Remove the hose clamp from the tubing connected to the culture vessel (1) and clamp-off the outlet tubing (5) now.

3.

Draw the plunger of the syringe (4). The sample is sucket through the sampling pipe and the tubing (1) into the sampling tube. Transfer the required sample volume. Then push the plunger back a little until the tubing connection to the culture vessel is empty. This avoids that residues of the sample are kept in the tubing until the next sample is taken.

4.

Losen the hose clamp from the outlet tubing and clamp-off again the transfer tubing from the culture vessel (1).

5.

Lead the outlet tubing (5) into a receptacle (flask, beaker, etc.). To transfer the sample slowly push the plunger of the syringe. The sample is driven through the rising pipe of the sample tube and the outlet tubing into the receptacle.

Notes:


It is recommended to completely empty the sample tube as shown in step 5. Sample residues in the tube may contaminate the next sample and change its characteristics.




Between two samples dip the open end of the hose into a disinfectant in order to avoid contamination of the culture broth.

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Operating Manual BIOSTAT B

Control Unit

5

CONTROL UNIT

5.1

General Information

The control unit of the laboratory scale fermentor BIOSTAT® B includes a digital measurement and control system. The section below describes all standard functions available with the current software version 4.15-.1). For a detailed technical information about the hardware design, the interface specifications and the default settings please refer to the supplement. The protocol for communication with a host computer is described in details in the manual „DCU-Host Communication“5-2). 5.1.1

Operating Behaviour

The measurement and control system stores all parameters which are adjustable by the operator (setpoints, calibration parameters, etc.) in a battery-buffered memory. These parameters will be backed-up when the BIOSTAT® B is switched-off and are available again when the fermentor is restarted for another operation sequence and after power failure. For the starting behavior of certain system functions which have direct access to the fermentor (some controllers, for instance) and for the outputs of the measurement and control system two different types of power failure are differentiated :


Switching the BIOSTAT® B „off“ and „on“ via the mains switch on the front panel.




Shut down caused by a power failure (mains cut-off).

5.1.2

Switching the BIOSTAT® B „Off“ and „On“

The BIOSTAT® B will be switched on or off, respectively, via the mains switch on the front panel of the control unit. After switching-on the measurement and control system is in this state of operation :
5.1.3

All controllers are switched off, actuators are in rest (neutral) position. Power Failure

In the menu „Maintenance“ a maximum power failure time (FAILTIME) can be set. See section „Utility Functions“, on page 5-37, for details. In the case of a power failure, after being restarted or when the mains supply is available again, the system continues all activities as follows:


At a power failure which is shorter than the time set for the FAILTIME all controllers will continue with the setpoint active before the power failure




If the power failure lasts longer than set by the FAILTIME, the measurement and control system will be switched over into a defined basic state (default state). It behaves as if being switched-off by the mains switch.

5-1)

If you have a control unit with another software version, please contact B. Braun Biotech International GmbH for a corresponding version of the Operating Manual.

5-2)

Further information available on request. Please contact your B. Braun representative or B. Braun Biotech International GmbH, see address in introductionary page.

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Operating Manual BIOSTAT B

Control Unit

5.1.4

Menu Structure

The operation display of the BIOSTAT® B is divided into the main function and function level: Main functions

Functions

Process Values

Process Values (display of process data in various forms)

Calibration

Calibration function for probes and pumps, as used for – pH - measurement and control – pO2 - measurement and control – ACID – pump – BASE – pump – foam measurement and control AFOAM – level measurement and control LEVEL – substrate control SUBS1 ... 2

Control Loops

DDC controller functions: – temperature control ”TEMP“ – stirrer speed control “STIRR“ – pH-control “pH“- pO2-Control “pO2“ – pump control function “AFOAM“- pump control function “LEVEL“ – pump control function “SUBS1 ... 2“ – gasmix control function “GASMX” – AIRFLOW - control (only with optional massflow control)

Maintenance

Function for system maintenance and trouble shooting; configuration of in/outputs, such as : – RECORDER – PRINTER – HOST – UTILITY – MEASUREMENT RANGES – manual operation (DIGITAL OUTPUTS) – manual operation (ANALOG OUTPUTS)

Sterilization

Function for culture vessel sterilization

Fig. 5-1:

Menu structure of the control system

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5.2

Operation of the Control Unit

5.2.1

General Operating Information

1.

You will select the required main function by pressing the corresponding main function key. The possible main functions are directly labelled on the keys. An active main function will be displayed in the first row of the LCD and indicated by a lighting LED in the key.

2.

Each main function except of „PROCESS VALUES“ has several subfunctions arranged in a defined order. As an example you'll always find the submenu „pH-Sensor“ at first whenever you select the main function „CALIBRATION“ or the submenu „Temp“ whenever you select the main function „CONTROL LOOPS“.




To switch over to subsequent menues of a main function repeatedly press the main function key. See above figure for the order of the submenues within each main function. You may change from one main function to another at any time by pressing its main function key.

3.

Non-numerical entries in a submenu are used for selecting the state of operation (such as switching over „auto“ „man“, „off“ „on“). To switch over to an alternate state of operation press the „ALTER“-key. Allways confirm the change of an entry by pressing „ENTER“. Otherwise the change will be ignored.

4.

If a menu includes more lines than visible on the actual display, either the top line or the lowest line of the information entry will include an arrow „ „ or „„. You can switch over to additional lines using the cursor-keys. Switching over usually takes place line-by-line, except with the Process Display, which displays a new page, then.

5.

The access to the menues for adjustment of system functions, like parametrization of the controllers, is protected by a password. Note the information about the available passwords in the corresponding sections below. When you enter a password into the corresponding entry, the entry will not be displayed.

6.

If it is difficult for you to read the LCD, you can change its contrast. This is done with the main function „MAINTENANCE“, in submenu „UTILITIES“.

Fig. 5-2: Operating terminal of the BIOSTAT® B measurement and control system

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5.2.2

Basic Menu Structure

1st line :


For main functions with several subfunctions (except PROCESS VALUES) the first line is a headline which identifies the selected function. Data may also be displayed in this line. The headline will always be displayed, even when you scroll down to following lines of the menu.




Different main functions may show the same headline. Note the LED's in the main function keys, the LED of the main function selected at this time will illuminate. Example : TEMP : 37.5 oC

2nd to 4th line :


Information entry, line - structure. In some cases, if the parameters cannot be displayed within the three lines available, you can scroll down to following lines. The headline will remain unchanged at this time. Example : pO2 SP MODE PARA

5.2.3

: 88,5 % : 83,4 % : auto :

 GASM

Operating Information

Symbol/entry

meaning, possible input

 (in top line)

Additional lines available before the actual line. – You can scroll upward to above lines with the „„-key.

(in lowest line)

Additional lines to follow within this menu. – You can scroll down to following lines with the „ „-key.

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5.3

Main Function „PROCESS VALUES“


The main function PROCESS VALUES only includes one function. The display has no headline. If you scroll down to lines beyond the 4th line even the top line will change. Operating display, page 1: TEMP : 42.7 oC STIRR: 800 rpm pH : 10.3 pH pO2 : 80.4 % Operating display, page 2: ACID : 32 BASE : 200 AFOAM: 12 LEVEL: 1300

ml ml ml ml

ñ

Operating display, page 3: SU1DC: 25 ml/m SUBS1: 3.0 % SU2DC: 25 ml/m SUBS2: 3.0 %

ñ

Optional process value display: GASMX: AIRFL: EXT_1: EXT_2:

25.00 l/m 0.0 % 0.0 %

ñ

additional pages of optional display EXT_3: EXT_4: FOAM : LEVEL:

0.0 % 0.0 % off off

ñ

HEATC: MFAIL: HIFOM:

off off off

ñ

Symbol / entry

meaning, possible input

TEMP, etc.

display of process value with physical unit, such as – process values of analog inputs (Temp, Stirr, etc.) – dosing counters – process values of digital inputs (Foam/Level)

,

Display of additional lines accessible via „ , „-keys

LEVEL

Dosing counter of level control pump

SU1DC

Dosing counter of substrate pump 1

SUBS1

Display of substrate setpoint of substrate pump 1

SU2DC

Dosing counter of substrate pump 2

SUBS2

Display of substrate setpoint of substrate pump 2

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Symbol / entry

meaning, possible input

GASMX

Display of setpoint of gasmix controller

AIRFL

Display of airflow rate

EXT_1 ... 4

Display of up to four input signals of remote devices: – the entry is in terms of „0 ... 100 %“ for input signals of „0 ... 10 V“ (or of 0/4 ... 20 mA, if inputs are set as current signals).

FOAM, LEVEL

Display of „foam“ or „level“ signal

HEATC

Display of heater monitoring; in case of malfunctioning the system displays an additional error message

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5 Control Unit

5.4

Main Function „CALIBRATION“

5.4.1

Calibration of the pH - Electrode

5.4.1.1

General

The pH-electrode is calibrated by a two-point calibration which determines the electrode parameters „zero drift“ and „slope“ using buffer solutions. The calibration is to be done before the electrode is installed in the culture vessel. For calibration you can use technical buffers, but we recommend that you should prefer original buffer solutions of the manufacturer of the electrode.


For the specifications and the assembly of the pH-electrode see section 4.

Warning: The calibration buffers can cause burns. Be careful when handling the buffers. You should wear protective gloves during the calibration procedure. The software of the control system calculates the pH-value from the electrode voltage with regard to „zero drift“ and „slope“, considering the Nernst equation. To compensate for effects of temperature the actual temperature can be entered manually during the calibration. The pH - electrode is calibrated before mounting into the culture vessel, i.e. prior to its sterilization. The high temperature during the sterilization can cause a shift of the electrodes’ zero. Also chemical compounds of the culture broth may effect the measuring characteristics of the electrode. Therefore the pH-electrode should be calibrated and checked for proper function regularly. To compensate for changes of the measurement characteristics of the pH-electrode the software offers a recalibration function. You can measure the pH-value externally in a sample taken from the process and enter this pH-value into the calibration menu. The system will then recalculate the calibration function and apply the new parameters for the pH-control function. For measuring the pH-value in such a sample, you should consider the following:


Use only fresh samples. Take care that the sampling method and the containers do not influence the pH-value of the sample




The devices used for external pH - measurement should ensure precise pH-measurement




If the pH - value measured by the BIOSTAT® B control system and the externally measured pH-value largerly deviate from each other, we recommend to check you remote measurement procedure twice rather than changing the calibration parameters. Entering of wrong recalibration parameters into the system can considerably influence the control behaviour and the supply of corrective solutions, which then will disturb your process.

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5.4.1.2

Operating Display : pH TEMP BUFZ BUFS

: : : :

7.30 23.4 7.00 4.01

pH o C pH pH

man ok ok

pH : 7.30 pH RECAL: 6.76 pH ZERO : 4 mV SLOPE: 58.4 mV

no ok ok

Symbol / entry

meaning, possible input

auto/man

When the menu is selected, the cursor is placed at this entry, showing the mode which has been selected previously. Press ALTER to change the temperature compensation mode: – auto: pH-measurement during the process; with temperature compensation using the measured temperature; – man: mode for pH-calibration, with temperature compensation by the temperature which is entered manually
change to „man“ mode for pH-calibration using the temperature labelled on the buffer bottle for the reference pH-value;

TEMP

display of culture vessel temperature, when in the „auto“ – mode input of the calibration temperature when in the „man“ - mode

BUFZ

input of the pH - value of the zero buffer (as labelled on the buffer bottle for the actual temperature)

ok

confirmation of the calculation of the zero point; after confirmation the cursor jumps to the BUFS - entry

BUFS

input of the pH - value of the slope buffer (as labelled on the buffer bottle for the actual temperature)

ok

confirmation of the calculation of the slope. After confirmation the cursor jumps to the „man/auto“ - entry. The temperature compensation will automatically switch over to „auto“ mode

RECAL

display and input of separately measured pH-value for the recalibration of the pH-electrode – no: the electrode will not be recalibrated – yes: the electrode will be recalibrated

no / yes ...

After pressing ENTER the cursor jumps to the „RECAL:“ entry. Here you can enter the pH-value measured in a sample taken from the process. After confirmation of the manually entered pH-value with ENTER the system adapts the „zero“ - calibration to the actual conditions ZERO

display of the electrodes’ zero in [mV]

SLOPE

display of the electrodes’ slope in [mV]

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5.4.1.3

pH - Calibration Routine

1.

Press the main function key „Calibration“ once to select the menu for pH-calibration. In the menu the cursor is on the „man/auto“ - entry.

2.

To allow for entering the reference temperature labelled on the buffer bottles for the relationship of pH versus temperature of the buffer press „ALTER“ to select „man“ual temperature compensation. Confirm with „ENTER“. The cursor jumps to the „TEMP“ - entry.

3.

Fill „zero buffer“ of pH 7.0 into a beaker. Place the pH-electrode into the beaker. Measure the temperature of the buffer and enter this as reference temperature into the TEMP entry. Confirm with „ENTER“. The cursor jumps to the „BUFZ“ - entry.

4.

Check the reference pH-value of the „zero“ buffer labelled on the bottle for the actual buffer temperature. Enter this pH-value in the BUFZ-entry and confirm with „ENTER“. The cursor then jumps to „ok“ and the system starts an automatic monitoring of the electrode. If the measured signal is constant within the given limits, the system stores the measured voltage as the electrode´s zero. Then the cursor automatically jumps to the BUFS entry.

5.

Fill some slope buffer of either pH 4.0 or pH 9.0 (depending on the expected pH-range during in the process) into a beaker. Remove the electrode from the beaker with zero buffer, flush with (demineralized) water and place into the beaker with the slope buffer.




Never dry the elektrode tip using a paper cloth. This may disturb the calibration.

6.

Check the reference pH-value of the „slope“ buffer labelled on the bottle for the actual buffer temperature. Enter this pH-value in the BUFS-entry and confirm with „ENTER“. The cursor then jumps to „ok“ and the system starts an automatic monitoring of the pH-electrode. If the measured signal is constant within the given limits, the system stores this as electrode slope.

7.

The cursor automatically jumps to the „man/auto“-entry and switches over the temperature compensation to „auto“-mode. For the pH-measurement the system calculates the pH-value from the electrode voltage using the Nernst equation with regard to the calibrated „zero drift“ and „slope“ and to the temperature measured in the culture vessel.




At step 4 and 6, when the cursor is on the „ok“ entry, you can shorten the automatic monitoring of the electrode. Wait about 10 seconds and press ENTER then. The system stores the actual signal as reference value of „zero“ and „slope“.

5.4.1.4

Notes on Recalibration of the pH-Electrode During the Process

1.

Use a fresh sample taken from the process in such a way, that its pH-value cannot be effected by the sampling procedure.

2.

Measure the pH-value using a precise pH measuring system. Enter the values into the corresponding entries of the pH-calibration menu, see description of the menu for details.




The „zero“ and „slope“ measured during the first calibration are displayed in the calibration menu until they were overwritten by manually entered values.




By comparing the pH-value measured in the process and the one measured in the sample you can evaluate the accuracy of the pH-electrode. This will also indicate whether the sterilization or the process conditions have changed the measuring characteristics of the electrode and whether the pH-electrode should be serviced or replaced.

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5.4.1.5

Notes about the Calibration Routine




The automatic monitoring of the electrode during the calibration routine shown above considers the following references and limits : – measured signal is stable within 0.2 % of the measurement range for 60 sec.; – voltage for electrode zero is kept within the range of -30 ... +30 mV; – electrode slope within the range of 54 ... 60 mV/pH




If the measured signal is outside these limits, the system displays an alarm message, such as „**ALARM**, Calibr. out of range, date time“. The calibration has failed and the pH cannot be measured correctly. In this case you should check whether : – all parts are properly connected; – you had followed the above calibration procedure accordingly.




If the error message appears again, the electrode must be serviced or replaced.




The pH-value displayed during the calibration procedure is invalid. The system will display the correct value after completion of the calibration course.

5.4.1.6

Special Notes on Handling of the pH-Electrode




The electrode has a restricted maximum lifetime which depends on the operating conditions and the application. The electrode should be serviced or replaced, whenever the functional check and the calibration indicate malfunctioning.




You'll find more detailed information about handling and mounting of the pH-electrode in section 4 of this manual. Also consider the documentation delivered with the electrode.

Note: Depending on the type of the delivered pH-electrode, the handling, operation and maintenance measures may differ from the information herein. You'll find more specific and detailed information about the electrode, the recommended service periods, the estimated lifetime and proper handling during calibration in the documentation of the manufacturer.

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5.4.2

Calibration of the pO2- Electrode

The pO2 - electrode is calibrated in percentage oxygen saturation, „% pO2“. The calibration is a twopoint calibration. Usually a measuring solution without oxygen gives the „zero“-current and a solution saturated with oxygen is defined as „100 % saturated“. The resulting electrode parameters „zero current“ and „slope“ are used to calculate a calibration function for pO2-measurement of the process. The pO2-electrode will be calibrated after mounting into the culture vessel and after the sterilization. For measurement of the electrode´s „zero“ the culture medium can be gassed with nitrogen to replace any oxygen dissolved in the medium. Supply of air or a specific gas mixture required for the process up to saturation of the medium with oxygen gives the electrode´s „slope“, 100 % pO2. The calibration menu for the pO2-electrode displays the actual „pO2“ as percentage saturation „% pO2“, the zero current and the slope. This allows a simple function control of the pO2-electrode. 5.4.2.1

Calibration Operating display pO2 TEMP NITR AIR

: 89.3 % : 23.4 degC : 0.0 % : 100 %

pO2 : 89.3 AIR : 100 ZERO : 3.6 SLOPE:160.3

% % nA nA

auto ok ok

ok

Symbol/entry

meaning, possible input

auto/man

When the menu is selected, the cursor is placed at this entry, showing the mode being previously selected; press ALTER to change the temperature compensation mode: – auto: temp. compensation using the measured temperature; – man: temp. compensation using the temperature which is entered manually

TEMP

Display of vessel temperature, when in the „auto“ - mode; input of the calibration temperature when in the „man“ - mode

NITR

Display of zero pO2;

ok

Confirmation of the calculation of the zero point; after confirmation the cursor jumps to the AIR - entry;

AIR

Display of the pO2 - value at optimum aeration of the culture medium for slope calculation

ok

Confirmation of the calculation of the slope. After confirmation the cursor jumps to the „auto/man“ - entry. The temperature compensation will automatically switch over to „auto“ mode

ZERO

Display of the electrodes’ zero in [nA]

SLOPE

Display of the electrodes’ slope in [nA]

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5.4.2.2

Course of the Calibration Routine




At a first use or at any time the pO2-electrode has been disconnected for more than 5 ... 10 min from the amplifier, it must be polarized. Polarization may take up to 6 h (shorter, only if the electrode has been disconnected for a few minutes).




In a culture vessel the pO2-electrode will be calibrated after the sterilization. Any interaction of this environment with the electrode, such as impacts of heat or chemical reactions of compounds of the culture medium with the membrane or the electrolyte will be considered for the calibration.




The electrode „zero“ can be calibrated when the sterilization cycle is switched over to COOL1-phase. Since the heating of the culture medium causes the dissolved gasses to be exhausted, the pO2 should be near zero, then. This state is identified as „0 % pO2“. Existing actual currencies measured by the electrode are within the accuracy of the measuring system. Therefor this method will be precise enough for most of the applications.




If a precise „zero“ measurement is required, the vessel can be gassed with nitrogen to replace any remaining dissolved oxygen. For this you can supply an oxygen-free gas (N2 of 99.8 % purity, for instance) and degas the dissolved oxygen from the culture medium.




The electrode „slope“ will be calibrated while the fermentor is at operation conditions (temperature, etc.) and the culture medium is saturated with oxygen.

Steps of Operation for Calibration : 1.

During the sterilization of the culture vessel wait until the BIOSTAT® B has switched to COOL1-phase. For „zero“ calibration using supply of oxygen-free gasses, wait until the measured pO2 is constant near „0 %p pO2“

2.

Press main function key „Calibration“ twice to enter the menu for pO2-calibration. The cursor will be in the „auto/man“ entry.

3.

If the mode is „man“ press ALTER to change into „auto“-mode for temperature compensation considering the actual temperature in the culture vessel. Confirm your selection with ENTER, the cursor jumps to the „NITR“ - entry.

4.

For zero calibration confirm „NITR : 0,0 %“ with ENTER. The cursor then jumps to „ok“.




The system starts an automatic monitoring of the electrode. After autoclaving and prior to supply of any gas containing oxygen the measured NITR-value should be near „0 % pO2“. and the electrode´s zero current should be about „0 nA“. If the measured signal is constant within the given limits, the system stores this as electrode zero. The cursor automatically jumps to the AIR entry.

5.

Prior to starting the fermentation run adjust the fermentation conditions and gas the culture vessel with air or the gas mixture provided for the process at the required flow rate. Confirm „AIR : 100 pO2“ with ENTER. If the slope reference should not be 100 % pO2, enter the intended value into the AIR entry and confirm a changed input with „ENTER“.




The cursor then jumps to „ok“. The system starts an automatic monitoring of the electrode. If the measured signal is constant within the given limits, the system stores this as slope. The cursor automatically jumps to the „auto“-entry.




During the process the system calculates the pO2-value from the electrode current with regard to the „zero“, „slope“ and the temperature. Since the calibration refers to the reference conditions (flow rate and type of gas) applied for the calibration, the actual pO2 measured can exceed 100 % O2, depending on the flow rate and kind of gas supplied.

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5.4.2.3

Notes about the Calibration Routine




The pO2 - measuring value displayed during the calibration course is invalid. The correct process value is only available after running the calibration.




The automatic monitoring of the electrode after confirmation of the NITR and AIR entries considers the following references and limits: – measured signal stable within 0.2 % of the measurement range for 60 sec.; – current for electrode zero is kept within the range of NITR 0 ... +15 nA; – electrode slope within the range of AIR: 25 ... 200 nA




If the measured signal is beyond these limits, the system displays an alarm message, such as „**ALARM**, Calibr. out of range, date time“. In this case the calibration routine has failed and the pO2 cannot be measured correctly. However, the system will accept the calibrated „zero“ and „slope“. You should repeat the calibration and check whether : – all parts are properly connected; – you had followed the above calibration procedure accordingly.




If the error message appears again, the electrode must be serviced or replaced




At step 4 and 6 of the calibration routine, when the cursor is on the „ok“ entry, you can shorten the automatic monitoring of the electrode. Wait about 10 seconds and press ENTER then. The system stores the actual signal as reference value of either zero and slope.

5.4.2.4

Special Notes on Handling of the pO2-Electrode




The pO2-electrode is subject to wear and tear, due to the impacts of heat during sterilization and due to possible chemical reactions of compounds of the medium with the membrane or the electrolyte. Such effects may alter the membrane structure, ingredients of the media may bind to the membrane or ions may permeate into the electrolyte. This will change the measurement characteristics more or less by time. Therefore the electrode has a restricted maximum lifetime which depends on the operating conditions and the application.




The electrode must be serviced whenever the electrode measures currencies exceeding the limits given above, while no oxygen is present, or the electrode shows a slow response.




The service of the pO2-electrode is restricted to – cleaning of the anode – replacement of the membrane module – refilling of fresh electrolyte




We recommend that you should refill with new electroly after about 5 times of sterilization of the entire unit or after about 6 weeks of use for measurement. Depending on physical/chemical properties of the culture solution it may be necessary to do this more often.




If refilling of new electrolyte cannot improve the measurement characteristic, the anode must be cleanend and/or the membrane module must be replaced. See the information concerning maintenance and service in the documentation of the electrode´s manufacturer.




You will find detailed information about mounting of the pO2-electrode in section 4 of this manual. However you should note, that the required operation and maintenance measures may differ from the information herein, depending on the type of the pO2-electrode delivered. Specific information about proper handling of the electrode, the recommended service periods, the estimated lifetime is available in the documentation of the manufacturer.

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5.4.3

Pump Calibration

For monitoring of the corrective solution consumption the measurement and control system can totalize the dosing times of the pumps and use them as process values. For this it converts the dosing times into delivered volumes with regard to the specific flowrates of the pumps. This is possible for the following pumps and operation modes :


st

1 pump: supply of an acid, mode „ACID“ nd 2 pump: supply of alkaline solution, mode „BASE“ rd 3 pump: for supply of antifoam agent, mode „AFOAM“ th 4 pump: substrate supply or harvest pump for level control, mode LEVEL th st 5 pump: 1 pump for substrate supply, mode „SUBS1“ nd 6th pump: 2 pump for substrate supply, mode „SUBS2“

The flowrates can be calculated automatically from the measured running time of the pumps and the delivery of the pumps during the calibration. The flowrates can also be entered directly via the operating terminal. You can zero the dosing counters for the pumps at any time via the operating terminal.
5.4.3.1

Since the calibration and dosing counter functions are the same for all pumps, only those for the acid pump will be described in details herein. Operating display ACID : 1280 ml MODE : calib TOTAL: 200 ml FLOW : 1.2 /m

strt

Symbol / entry

meaning, possible input

ACID

Display of acid volume delivered; the counter will be set to zero : – at switching on the unit with the mains switch; – when switching the operation mode over from „calib“ to „total“


for the other pumps instead of ACID the following entries are shown : BASE

Display of delivered volume of alkaline agent („BASE“)

AFOAM

Display of delivered volume of antifoam agent „AFOAM“

LEVEL

Display of actual value of the LEVEL dosing counter

SU1DC/SU2DC

Display of the substrate volume delivered by pump SUBS1 or SUBS2

MODE

Enter mode of the dosing counter : – calib: calibration of flow rate (delivery) of the pump; – total: dosing counter active;switching from „calib“ to „total“ mode sets the counter to zero.

strt/stop

Enter operation mode „start/stop“ of the pump calibration routine. This entry is only active in the „calib“-mode.

TOTAL

When switched into „calib“ - mode, the delivered volume can be entered herein

FLOW

Display of actual pump delivery or input of delivery rate. It is possible to enter a delivery rate manually at any time

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5.4.3.2

Calibration of a Pump Installed in the Control unit

1.

Place a piece of tubing of suitable length with one end into a beaker filled with water, mount the tubing into the pump head and place the outlet end into a measuring beaker. The tubing must have the same size as used for medium transfer (such as 1.6 x 1.6 or 3.2 x 1.6 mm).

2.

To avoid that the clearance volume of the tubing is considered for calibration (this will cause a calibration error) activate the pump manually until the tubing is completely filled.

3.

Press the main function key „Calibration“ several times to select the menu of the pump which should be calibrated (ACID, BASE, AFOAM or LEVEL). Move the cursor to line „MODE : total“. Press the ALTER-key once to change to „MODE : calib“. Confirm with „ENTER“. Then the cursor jumps to „strt“.

4.

Press „ENTER“ to start the pump for calibration. At the same time the entry at „TOTAL“ will be deleted. The pump delivers liquid for a certain period. The system automatically switches over to „stop“ while the cursor remains in this entry. Confirm „stop“ with „ENTER“. Then the cursor jumps tho „TOTAL“.

5.

Measure the volume delivered into the measuring beaker and enter the measured volume in entry „TOTAL“. Confirm your input with „ENTER“.




Now the system calculates the flowrate, which will then be displayed. Confirm with „ENTER“ as well. The cursor jumps to entry „MODE“. The operation mode automatically changes from „calib“ to „total“. The existing entry of „TOTAL“ will be deleted.




If you want to calibrate another pump, select the corresponding menu using the main function key „Calibration“ and repeat the steps shown above.

5.4.3.3

Special Notes




You must use tubings of the same size for calibration and delivering the addition solution (tubing size Ø 1.6 x 1.6 or 3.2 x 1.6 mm, for instance). Else the system will calculate the dosage using wrong calibration parameters.




You can do the calibration before start-up and sterilization of the corresponding storage supplies. However, recalibration is possible at any time using tubings of the same size as connected to the storage bottles.




The dosing counter will be set to zero when the control unit is switched-off. When disabling the controller with „stop“ the dosing counter will not be set to zero but continue with the actual value after restart.




During a fermentation run you should handle the assemblies with special care to prevent unintentional loosening of the connections to the culture vessel or damages of the tubings.

Caution: Acid or akaline agent used for pH-control can cause burns, when the tubings get loose and the agents are released unintentionally.


Damages of the tubings can cause the corrective solutions to be contaminated by environmental germs. This may result in infections of the culture.

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5.5

Main Function „Control Loops“

All control loops in the measurement and control system system operate as DDC controllers. The controllers are implemented as PID controllers, setpoint controllers or on/off controllers and adapted to the control loops concerned. Depending on the connected actuators the control output is either continuous or pulsewidth-modulated. Both single- and split-range control operation are possible. The controller structure (P-I-D) can be parametrized according to the task, if necessary. The controller functions possible with the BIOSTAT® B are shown below. The controllers can be switched over to different modes of operation. Switching between the operating modes is carried out with bumpless transfer.


OFF

:

controller switched off with defined output




AUTO

:

controller in operation




CASC

:

controller operating as servo controller in cascade mode

In the operating display you can enter the actual value and the operating mode. Controller parametrization is accessible by entering the required password in the „PARAM“ - entry. Then the parametrization menu appears for adjustment of PID parameters and deadband, for instance. The BIOSTAT® B can be connected to a host computer and hence operate in remote operation mode. Setpoints and operating modes of the individual controllers are then determined by the host computer. The BIOSTAT® B operating terminal will be locked against inputs, then. 5.5.1

Extent of Controller Functions of the BIOSTAT® B Temp

PID cascade controller with pwm-splitrange outputs for heating via steam supply or electric heater and cooling via cooling water supply

Stirr

PID controller with analog output for motor control

pH

PID controller with pwm-split-range outputs for the acid & base pump

pO2

PID cascade controller for one or two servo controllers. Servo controller(s) can be – STIRR – AIRFL – Gasmix – SUBS1

FOAM

On/off (cycle/delay time) controller for antifoam

LEVEL

On/off (cycle/delay time) controller for harvest pump

SUBS1/SUBS2

Setpoint controller for external continuous substrate pumps or, as an alternative, for an internal pump, which has no control function (ACID/BASE or AFOAM/LEVEL)

Gasmix

Setpoint controller for pulse-width modulated splitrange outputs for N2 and O2 of a separate Gasmix unit (optional equipment)

Airflow controller

Setpoint controller for a massflow controller

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5.5.2

Controller Operation in General

All DDC controllers are operated in almost the same way. The „Control Loops“ menu of each of the controllers has two different displays:


The settings for the setpoints and the operating modes of the controllers are done in the „Operating Displays“, which are directly accessible. If more than 3 lines are required for displaying the settings of a controller, you can scroll up and down to following lines showing additional parameters using the cursor keys „ , “.




Any settings not required for routine operation, will be made using the parametrization functions.. The „Parametrization Displays“ are only accessible via a two-digit password The second page is accessible via a password.

5.5.2.1
5.5.2.2


5.5.3

Setpoint Adjustment Setpoints are entered and displayed in the SETP entry. Entered values must be within the measuring range of the variable. Values exceeding the measuring range will be ignored. Operating Modes Operating modes are entered and displayed in the MODE - entry. Modes are selected via the ALTER - key. Some controllers only allow specific operating modes. If a selected mode is not plausible for the controller, the inputs will be ignored. – In „off“ - mode the controller is inactive; the output adapts to a defined position (corresponding to the neutral position of the actuator). – In „auto“ mode the controller is active. Setpoint values can be altered via SETP. – In „casc“ mode the controller operates as a servo controller in a cascade control loop. The setpoint value is specified by the master controller; it cannot be adjusted via SETP. Parametrization of Controllers in General

On delivery the DDC-controllers are configured with such parameters, which ensure a stable operation of the controls for the individual type of fermentor. The default settings of the BIOSTAT® B are given in section “Factory Settings for Controller Parameters“ on page 5-42. If the settings are different for customized versions of the fermentor system, the corresponding documentation will be attached to this manual or be delivered extra. Note: Under normal circumstances is not necessary to change the control parameters. An exception are those control loops which are strongly influenced by the process, such as pH and pO2 control. If necessary for improved adaptation of the DDC controllers to the control loops of the fermentor, you may change these control parameters via the parametrization display of the corresponding controller :


DEADB

:

Deadband adjustment (only for PID controllers)




XP, TI, TD

:

PID-Parameter (only PID controllers)




MIN, MAX

:

Controller output limits

Within a controller display the parametrization display is only accessible via the required password. Note: As the passwords are shown in the corresponding sections of this manual below, the manual should only be available for personnel, which is qualified and authorized to enter the parametrization displays of the controllers and change the settings.


Inadequate changes of parameters can cause malfunctioning of the control system.

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5.5.3.1

Deadband

A deadband can be adjusted for PID controllers. If actual values vary stochastically a predefined deadband allows for more stable control at minimum actuator changes. The controller output remains constant or is set to zero (i.e. a specific feature of pH - controller) if the control deviation is within this deadband. In the case of splitrange controller outputs, a deadband can avoid oscillating of the output (which may result in a continuosly alternating acid-base titration by the pH-controller, for instance).


The deadband is adjusted in the entry DEADB, it is the percentage [%] of the measurement span of the actual value and is symmetrical to the adjusted setpoint.




Example for pH control: – measurement range pH = 2 - 12 pH, measurement span 10 pH – adjusted deadband 1% = ± 0.1 pH, adjusted setpoint 6.0 pH




Here the control is inactive at an actual value within 5.9 pH - 6.1 pH. The acid pump will be activated when the actual value exceeds the upper limit, the alkaline agent pump will be activated when the actual value falls below the lower limit.

5.5.3.2

PID - Parameters

The PID controller can be optimized via the following PID parameters : XP

Proportional range in % of measurement range (P-section)

TI

Reset time in seconds (I-section)

TD

Rate time in seconds (D-section)

The PID parameters can be adjusted in the corresponding entries. The digital controller implemented operates according to the positioning method. Setting individual PID parameters to zero determines the control principle. For easy optimization of the controllers you can change the parameters during operation. Set the parameters for the controller functions as follows:

5.5.4

to work as

set the PID – parameters as follows

P-controller

TI = 0, TD = 0

PI-controller

TD = 0

PD-controller

TI = 0

PID-controller

all PID - parameters have defined set values

PID - Controller Optimization

You will need some detailed knowledge about the control theory for optimum adaptation of a PID controller to the corresponding control loop. For detailed information about proven adjustment rules you may refer to any standard literature. Therefore the following specifications are only general guidlines:


The D section (TD) should only be activated in the case of rather stable actual values since the controller output will change considerably and fast if the actual values are varying stochastically. Else this would lead to an unstable control.




As a general rule the TI : TD ratio should be 4 : 1.




In the case of a periodically oscillating control loop you can increase Xp and/or increase TI/TD to minimize or even prevent oxcillation.




If an actual value adapts to the setpoint to slowly after a setpoint jump or if the actual value shows a continous change (drift), you can decrease Xp and/or decrease TI/TD

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5.5.5

Temperature Control

5.5.5.1

Functional Principle

The temperature controller is a single-loop PID controller. The vessel temperature is the reference input. The controller output controls the cooling valve and the heater in split-range operation via pulsewidth-modulated outputs. As a special feature, the master controller can be switched from PD (which is the start-up-mode) to PID - operation when approaching the setpoint. Thus overshooting is eliminated as far as possible. When the temperature controller is switched off, an additional digital output also switches off the circulation pump and the heating contactor in the fermentor. 5.5.5.2

Operating Information Operating display page 1 : TEMP SETP MODE PARA

: : : :

37.5 oC 33.4 oC auto _

Symbol / entry

meaning, possible input

TEMP

Display of actual value

SETP

Display or input of controller setpoint

MODE

Display and input of control mode: – auto: automatic operation according to given setpoint; – off: controller is switched off, outputs are in operating state

PARAM

Enter 2-digit passwort „19“ for access to the controller parameters. When entering the password it will not be shown in the display.

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Operating display page 2, with cursor within the upper 4 lines :

TEMP MIN MAX XP

: 37.5 oC : -100 % : 100 % : 10.0 %



Operating display page 2, with cursor moved down to the lowest line :

TEMP : 37.5 oC TI : 300 s TD : 75 s DEADB: 00.0 %



Symbol / entry

meaning, possible input

MIN

Display and input of minimum controller output limit. For the temperature controller this parameter limits the output for „cooling“

MAX

Display and input of maximum controller output limit. For the temperature controller this parameter limits the output for „heating“ Note : The values for „MIN“ and „MAX“ depend on the individual measurement range. If the measurement ranges are changed, the „min“ and „max“ values for the outputs are changed correspondingly and cannot be exceeded

XP

Enter proportional range

TI

Enter integral portion (reset time)

TD

Enter differential portion (delay time)

DEADB

Enter dead band

,

More lines are available via the „ , “ - keys

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5.5.6

Stirrer Speed Control

The stirrer speed control function works as a PID controller. It sets the stirrer speed, supplies the digital setpoint signal for the motor control and displays the input signal from the motor control. When the controller is off, the motor contactor is switched off via a digital output. In conjunction with the pO2controller the stirrer speed controller can be servo controller in the pO2 - cascade control loop.


The stirring speed is adjustable in the range of 50 ... 1,200 rpm.




The stirring speed must be limitted to max. 600 rpm for 10 l vessels and to about 300 rpm for vessels with a silicone membrane insert. Higher speeds may cause inadmissible vibrations of the assembly of culture vessel with motor or can result in damages of devices inside the vessel (i.e. loosening of the silicone membrane).




A system reset will enable the default speed settings. Therefore, after a system reset the speed limit settings should be checked and the max. speed be reconfigered. Operating display: STIRR: 750 rpm SETP : 750 rpm MODE : auto PARA : Symbol / entry

meaning, possible input

STIRR

Display of actual value

SETP

Display or input of controller setpoint

MODE

Display and input of control mode: – auto: automatic operation at given setpoint – casc: cascade operation. The controller is servo controller and triggers the output of the master controller; – off: the controller is switched off, outputs are in operation state

PARAM

Enter 2-digit passwort „19“ for access to the controller parameters

Operating display page 2, with cursor within the upper 4 lines : STIRR: MIN : MAX : XP :

750 rpm 004.2 % 100.0 % 200.0 %



Operating display page 2, with cursor moved down to the lowest line : STIRR: 750 TI : 5 TD : 0 DEADB: 000.0

rpm s s %



Symbol/entry

meaning, possible input

MIN, MAX

For limitting controller output to minimum or to maximum

XP

Enter proportional range

TI

Enter integral portion (reset time)

TD

Enter differential portion (delay time)

DEADB

Enter dead band

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5.5.7

pH - Control

The pH - controller of the BIOSTAT® B is a software function which acts as a PID controller. It triggers the corrective solution pumps for supply of acid and alcaline agent in split-range mode via two pulsewidth-modulated outputs. This allows for a reversible control of both pumps. If gaseous CO2 should be applied for pH - control instead of acids (as often applied in tissue cell culture), the output of the controller can be connected to external Gas Mixing Unit including a CO2 - control valve. The acid and base signals will remain switched-off as long as the deviation remains within the parametrized dead bands. This will prevent unnecessary supply of acid or base to the culture broth and will limit the consumption of these corrective solutions (this is also true for CO2, if applied). 5.5.7.1

Setting the Setpoint and Controller Mode Operating Display : pH SETP MODE PUMP

: : : :

7.25 pH 7.10 pH auto ACID/BASE

Operating display page 2: pH : 7.25 pH MODE ; auto PUMP : ACID/BASE PARAM:

Symbol / entry

meaning, possible input

pH

Display of actual value in the headline

SETP

Display or input of controller setpoint

MODE

Display and input of control mode: – auto: automatic operation with the given setpoint; – off: controller is switched-off; outputs are in the operation state

PUMP

Display and selection of the required pump(s) – ACID/BASE: both acid and base pump are operated – ACID/-----: the acid pump is operated but not the base pump; thus the base pump is free for use as the substrate pump – -----/BASE: the base pump is operated but not the acid pump; thus the acid pump is free for use as the substrate pump

PARAM

Enter 2-digit password „19“ for access to controller parameters; during entry the password will not be displayed

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5.5.7.2

Parametrization Parametrization display, with cursor within the upper 4 lines : pH MIN MAX XP

: 7.25 pH : -100 % : 100 % : 30.0 %



Parametrization display page 2, with cursor moved down to the lowest line: pH : 7.25 pH TI : 30 s TD : 0 s DEADB: 0.5 %



Symbol / entry

meaning, possible input

MIN

Display and input of minimum controller output limit. – At the pH controller this parameter limits the output for the acid pump

MAX

Display and input of maximum controller output limit. – At the pH controller this parameter limits the output for the base pump

Note :

The „MIN“ and „MAX“ values depend on the individual measurement range. If the measurement ranges are changed, the „min“ and „max“ values for the outputs are changed correspondingly and cannot be exceeded

XP

Input of proportional range

TI

Input of integral portion (reset time)

TD

Input of differential portion (delay time)

DEADB

Input of dead band

,

More lines are available below; move over using the „ , “ - keys

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5.5.8

pO2 - Control

5.5.8.1

Modes of Operation

The control unit of the BIOSTAT® B includes a pO2-controller for universal use. The pO2 control function can be applied as required for the intended process. You can adapt the pO2 control to even specific process requirements. In particular the following control strategies can be performed :


pO2-control by operating a single process value. These servo controllers can be selected: – stirring speed (STIRR) – substrate supply (SUBS) – gas supply by mixing of 2 gasses (GASMIX) – gas supply using the optional massflow controller (AIRFL)




pO2-control by operating two process values. The same servo controllers can be selected.

5.5.8.2

pO2 - Controller with One Servo Controller

In the pO2 control using one servo controller the pO2 controller acts as the master controller. Its output directly operates the input of the servo controller. In standard configurations the pO2-controller operates either the stirrer speed controller, the substrate supply or the Gasmix controller, respectively. By use of the Gasmix servo controller two gasses can be mixed, such as N2 and O2, for instance. For this the Gasmix controller operates a valve for O2 supply and a valve for supply of O2 in splitrange mode via pulse-width modulated outputs. This method is often applied for bubble free gas supply to the culture medium via a membrane aeration system. An alternative to the use of the Gasmix controller is the “O2-enrichement” operation. The pO2 controller then triggers a 3/2-way valve of the air (gas) inlet, where one input is connected to the air supply and the other input is connected to an O2 – supply. You will find an example for this further below. 5.5.8.3

pO2 - Controller with Two Servo Controllers

For the pO2-cascade control serial operation can be selected. Then the pO2-controller operates two servo controllers one after another, according to their priority. In the pO2-controller you can predefine a „min/max“ range for each servo controller. The pO2-controller triggers this servo controller as long as the related setpoint is within its „min/max“ range. If you set the reverse „min/max“ - settings you will get a reverse control.


When the pO2-control is switched on, the output of the pO2-controller operates the servo controller 1 (CASC1) with the given setpoints within the range of the preset „min/max“ settings. If selected, the servo controller 2 receives the „MIN“ setpoint of the pO2-controller.




At increasing O2 consumption, when the setpoint signal reaches the „MAX“ value defined for the servo controller 1, the output of the pO2-controller will automatically switch over to the setpoint input of servo controller 2. The delay time for swiching over is 5 min (default setting) and can be modified. Then the following setpoints will be transmitted: – predifined MAX output for servo controller 1 – controlled output of pO2-controller for servo controller 2




If the O2-consumption is reduced again, the servo controller 2 receives the setpoint signal defined in the pO2 controller as “MIN” and the output of the pO2-controller again triggers the servo controller 1.

This control principle allows for an accurate pO2-control during the process, the pO2 can be kept constant even at larger variations of the O2-consumption without any manual control required. Above this the PID-parameters of the servo controllers 1 and 2 can be parametrized independently from each other to provide an optimum adaptation of the controls to the behaviour of the control system.

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5.5.8.4

Operating Information Operating display, cursor in the first line: pO2 SETP MODE CASC

: 88.5 % : 83.4 % : auto :STIRR

AIRFL

Operating display, cursor in the last line: pO2 : 88.5 % MODE : auto CASC :STIRR PARAM:

AIRFL

Switching over to cascade operation with one servo controller: pO2 : 88.5 % MODE : auto CASC :STIRR PARAM:__

STIRR

Symbol / entry

meaning, possible input

pO2

display of actual value in the headline

SETP

display or input of controller setpoint

MODE

possible modes are: – auto: automatic operation with the given setpoint; – off: controller is switched-off; outputs are in the operation state.

CASC

display or selection of servo controller 1 and, if necessary, servo controller 2 (the above example shows STIRR selected as servo controller 1 and AIRFL selected as servo controller 2) STIRR : output of the pO2-controller triggers the setpoint of the stirrer controller; the operating mode of the stirrer speed controller will automatically be switched to „casc“ -mode. SUBS : output of the pO2- controller triggers the setpoint of the substrate 1 controller; the operating mode for the substrate 1 controller will automatically be switched to „casc“ -mode GASM : output of the pO2-controller triggers the setpoint of the gasmix controller and the operating mode will automatically be switched to „casc“ -mode. AIRFL : output of the pO2 controller operates the setpoint of the airflow controller; the operating mode will automatically be switched to „casc“ -mode

Note :

For switching over to operation with one servo controller place the cursor at the tag of servo controller 2 and select the same controller as servo controller 1 via „Alter“; – confirmation with ENTER deletes the tag of servo controller 2.

PARAM

Enter 2-digit passwort „19“ for access to controller parameters

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Parametrization display, page 1, with cursor within the upper 4 lines : pO2 : 88,4 % DEADB: 0.5 % HTIME: 5 min STIR MIN : 0 %

ñ

Parametrization display, page 1, with cursor in the lowest line : pO2 MAX XP TI

: 88,4 % : 100 % : 100 s : 100 s

ñ

Parametrization display, page 2, with cursor in the lowest line : pO2 XP TI TD

: 88,4 % : 100 s : 100 s : 0 s

ñ

Symbol / entry

meaning, possible input

DEADB

Enter dead band

HTIME

display or input of the delay time for switching over from cascade 1 to cascade 2

STIR, etc.

selection of servo controller using the ALTER key STIR : display / setting of the controller parameters for the stirrer controller while used as servo controller

SUBS : display / setting of the controller parameters for the substrat controller while used as servo controller GASM : display / setting of the controller parameters for the gasmix controller while used as servo controller AIRFL : Display and adjustment of the controller parameters related to the servo controller „Airflow“, if this servo controller is available (Option) XP

Enter proportional range

TI

Enter integral portion (reset time)

TD

Enter differential portion (delay time)

, 5.5.8.5


More lines are available below; move over using „ , “ - keys

Special Notes For the pO2-controller two sets of parameters are internally available. Access is only possible to the PID - set, which has been activated before within „Mode“.

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5.5.9

Foam Control

5.5.9.1

Function Principles

When foam has contact with the foam sensor for longer than 6 seconds the foam amplifier releases a limit value signal, which is the input signal for the foam controller. The sensitivity of the foam measurement amplifier can be adjusted in the display of the foam controller. The delay time prevents erroneous activation of the foam control and supply of antifoam agent caused by short time contact or splashes of the liquid against the probe. The controller output operates the internal „Antifoam“ pump. The pump is switched-on and off periodically as long as foam is detected (sensor signal = on). Running time and the cycle time (= dosing time + delay time) for intermittent operation can be entered via the controller display. The amount of antifoam agent delivered by the pump depends on the hose dimensions. After pump calibration via the corresponding calibration function a dosing counter displays the delivered volume. The internal antifoam pump of the BIOSTAT® B can be used as substrate pump, if the foam control function is not required. 5.5.9.2

Operation of the Foam Control Operating Display, page 1: FOAM : off MODE : auto CYCLE:01:30 m:s PULSE:00:05 m:s

chg

Operating Display, page 2, with cursor in the lowest line: FOAM : off SENSI: 3 PULSE:00:05 m:s PUMP : AFOAM

ñ

Symbol / entry

meaning, possible input

FOAM

Display of actual sensor signal FOAM in the headline; – the tag FOAM will automatically be switched over to SUBS according to the preselected function of the controller

MODE

Enter controller mode : – auto: controller is active, automatic operation; – off: controller is switched-off; outputs are in the operation state; 1. Place cursor at „auto“ or „off“, respectively 2. Press ALTER to switch over the mode and confirm with ENTER

CYCLE

Enter cycle time in „Minutes:Seconds“ [mm:ss]

PULSE

Enter pump running time in „Minutes:Seconds“ [mm:ss]

SENSI

Enter probe sensitity; 4 levels of sensitivity are adjustable

PUMP

Display and selection of the pump connected to the controller: – AFOAM: the antifoam controller operates the antifoam pump – -----: the antifoam controller does not operate the antifoam pump; the antifoam pump can now be used for substrate supply.

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5.5.9.3

Special Notes




With the BIOSTAT® B chemical foam control using commercially available antifoam agents (such as greases, oils, oil-water-emulsions, silicone-oils, paraffines) is applied. You should note that these agents can affect the growth of the cells and cell metabolism. Therefore the amount of agent supplied to a culture vessel should be minimized.




Dosing of antifoam agent should only be activated, when foam has contact with the probe. Adjust sensitivity of the probe in that way, that splashing of bubbles, foam residues or culture medium against the probe will not activate the controller. Adjust cycle time long enough to differentiate between splashing or short contact of foam or medium caused by stirring and/or aeration and longer contact with foam.




The reference potential for the foam electrode is provided via the Pt-100. Thus the foam measuring system only works, if both the foam electrode and the Pt-100 are mounted into the top-plate of the culture vessel.

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5 Control Unit

5.5.10

Level Controller

Taking samples during a fermentation run will reduce the filling volume of the culture vessel. Therefore you may need to add medium the culture vessel regularly. You will also supply medium or specific substrates for fed-batch or continuous processes. This can cause that the maximum working volume of the vessel is exceeded and the surplus volume needs to be removed. The level control function of the BIOSTAT® B provides a monitoring of the filling volume of the culture vessel. In addition you can automate the removal of surplus medium as well as the supply of medium and specific substrates, in order to keep the filling volume in the culture vessel constant. 5.5.10.1 Function Principles of Level Control The insertion depth of the level probe determines the controlled filling volume of the culture vessel. Therefore you will need to gauge the capacity of the vessel and to determine the insertion depth of the probe. When the culture medium comes into contact with the probe, the level amplifier generates a digital signal, which is the input signal for the level control function. You can adjust the sensitivity of the amplifier to consider the conductivity of the medium and to avoid unintended controller activities due to contact of foam with the probe or by splashing of liquid against the probe, for instance. The level amplifier offers the two modes „HARV“ and „FEED“. In the „HARV“ mode the level controller output operates the „LEVEL“ pump, as long as the level signal is „on“. The pump acts as harvest pump. In the FEED - mode, it operates the pump, when the level signal is „off“. Then the pump acts as dosing pump. The pump operates at running and delay times, which can be adjusted in the corresponding operating display. The amount of medium which is removed or supplied, depends on the running time and the hose dimensions. If the pump is calibrated using the corresponding pump calibration function, the dosing counter displays the exact delivery volumes. 5.5.10.2 Operating Information


You will operate the level controller in the same way as the antifoam controller. See the above section for details.

5.5.10.3 Additional Notes


For a precise level control at certain filling volume you´ll need to adjust the level probe at the required height. For this you'll have to measure the vessel capacity at a given assembly of internal vessel component parts, such as inserts, stirrers, electrodes, etc.




You may label the intended filling volumes on the vessel. However, you should note, that high stirring speeds and/or intentisified gassing of the culture can produce a whirl or an increased level of the culture medium, compared to the level without or at low stirring speed or gas supply, respectively.

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5.5.11

Controller for Substrate Feed SUBS1 und SUBS2

The two controllers support external continuous pumps intended for substrate feeding. The controllers does not use feedback of the actual speed of the pump. The display of the process value „SUBS1“ and “SUBS2” represents the controller´s setpoint. The amount of substrate supplied with the pump depends on hose dimensions and running time. If the pump is calibrated using the pump calibration function (refer to the pump calibration instructions), the dosing counters SU1DC and SU2DC display the exact deliveries. You can switch the substrate controller to an internal peristaltic pump, if no external continuous pump is available but one of the internal pumps is (if unused). For switching over proceed as follows: 1. Clear the unused pump in the operating display of the controller assigned to it. 2. Assign this pump to the substrate controller. You can operate the substrate controller using a timer setpoint profile. Such a profile can have step changes and ramp changes with max. 10 segments and be started and stopped at any time. 5.5.11.1 Operation of the Substrate Supply (Example for SUBS1) Operating display SUBS1: 20 % SETP : 020.0 % MODE : auto PUMP : SUBS1 Operating display with cursor in the lowest line: SUBS1: MODE : PUMP : PARAM:

20.0 % auto SUBS1 __

ñ

Symbol / entry

meaning, possible input

SUBS1 / 2

Display of setpoint of substrate controller SUBS1 (/ 2, if applied)

SETP

Display or input of the setpoint for the pump delivery (flow rate)

MODE

Enter controller mode : – auto: automatic operation with the given setpoint; – off: controller is switched-off; outputs are in the operation state; – casc: substrate controller is switched to cascade operation for the pO2-controller; i.e. the pO2-controller delivers the setpoint for the substrate controller – profil: automatic operation using a defined setpoint profile

PUMP

Display and selection of the connected pump : – SUBS: the controller operates an external continuous pump; – ACID: the substrate controller operates the ACID pump, i.e. the pump is not used by the pH-controller; – BASE: the substrate controller operates the BASE pump, i.e. the pump is not used by the pH-controller; – AFOAM: the substrate controller operates the AFOAM pump, i.e.the pump is not used by the AFOAM-controller; – LEVEL: the substrate controller operates the LEVEL pump, i.e. the pump is not used by the LEVEL -controller

PARAM

Access to the controller parameters via 2-digit password „19“

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5.5.11.2 Parametrization of the Substrate Dosing Function SUBS1, Input of a Setpoint Profile Parametrization display page 1 SUBS1: 15.0 MIN : 000 MAX : 100 TIME0: 0:00

% % % SP :

10 %

1:10 2:00 3:15 4:00

SP SP SP SP

: : : :

15 20 35 40

% ñ % % %_

4:50 5:30 6:00 6:15

SP SP SP SP

: : : :

55 70 80 90

% ñ % % %

Parametrization display page 2 TIME1: TIME2: TIME3: TIME4: Parametrization display page 3 TIME5: TIME6: TIME7: TIME8: Parametrization display page 4 TIME9: 7:00

SP :

90 % ñ

Symbol / entry

meaning, possible input

MIN

Display and input of the minimum controller output limit

MAX

Display and input of the maximum controller output limit

TIME0...9

Display and input of the time for max. 10 profile changes

SP

Display and input of the setpoints for the profile changes

The above inputs will create this setpoint profile

Additional notes


If the time for the first breakpoint is not set to „00:00 h:m“ the system uses the current setpoint for the start of a profile.




In a program for a setpoint jump (step change) both setpoints can have the same time.




When a profile is defined and started for pO2-control, correlating active profiles for „SUBS1“ will be stopped automatically. The controller will be switched over to „cascade“-mode.

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5.5.12

Airflow Controller

5.5.12.1 Modes of Operation and Equipment Airflow controllers allow an automatic continuous control of the air supply according to the oxygen consumption in the process. The measurement and control system triggers the massflow controller via an analog setpoint signal. The airflow controllers is a massflow controller module integrated in the inlet air line behind the flowmeter, see P&I – diagram for details.


Standard equipment is a massflow - controller for air supply rates up to 10 l/min




Depending on the size of the culture vessel and the intended intensity of aeration different airflow controllers can be applied

5.5.12.2 Operating Information Operating display : AIRFL: 8.00 slpm SETP : 10.00 slpm MODE : auto PARAM:_ Parametrization display : AIRFL: MIN : MAX :

8.00 slpm 0 % 100 %

Symbol / entry

meaning, possible input

AIRFL

display of actual flowrate

SETP

entry of setpoint for the flowrate

MODE

enter controller mode : – auto: automatic operation with the given setpoint; – off: controller is switched-off; outputs are in the operation state;.

PARAM

access to controller parameter, enter 2-digit password „19“ for this

MIN

„min“ limit of controller output

MAX

„max“ limit of controller output

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5.6

Main function "Maintenance"

5.6.1

Recorders

5.6.1.1

Technical Set-up

Two types of recorders are available as standard equipment of BIOSTAT® B - fermentors:


Arucomp SK 10 recorder with digital display, Hartmann und Braun




Arucomp SK 10 recorder with analog display, Hartmann und Braun

5.6.1.2

Operating the Recorder




The recorder is contained in an extra housing and will be connected to the corresponding connector „Recorder“ of the control unit.




Below you will find an overview on the most important (default) adjustments for connection to a BIOSTAT® B. Additional information about individual functions and the adjustment of op5-3) eration parameters can be found in the Operating Manual of the recorder.

5.6.1.3

Recorder Configuration

The control unit includes a recorder output board with plug connection. This board offers 6 analog outputs 0 ... 10 V for the connection of a 6-channel dot printer. It is possible to assign up to 6 process values to the 6 recorder outputs. The signal ranges of the outputs are fixed and correspond with measurement range for the process values. Operating display page 1: Recorder CHAN1:TEMP CHAN2:STIR CHAN3:pH Operating display page 2, with the cursor in the lowest line : Recorder CHAN4:pO2

Symbol / entry

meaning, possible input

CHAN1

Enter process value for recorder output channel 1. – Select other process values with the „ALTER“ key. – Confirm selection with ENTER. – Moving on with a cursor key without ENTER will ignore your selection

CHAN2..4

see CHAN1

,

5-3)



More lines are available. Move over using „ , “

Included in the documentation set if part of the system configuration on delivery. Otherwise available on request. Please contact B. Braun Biotech International GmbH for this.

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5.6.2

Printers

5.6.2.1

Technical Set-up

Connection of a protocol printer to the BIOSTAT® B allows for easy online - recording of the running process. The protocol is a „hardcopy“ of the course of the fermentation run. Thus the course of the important parameters can be monitored and/or unexpected events can easily be detected, for instance. The printer can be a standard matrix printer with serial interfaces and small fonts. It will print the parameters numerically into a corresponding table. The protocol will include the data of the PROCESS DISPLAY and the following parameters (see example of a protocol below) :


Date, time of the print (showing the time of an event during the fermentation run)




All process values




Alarm messages

5.6.2.2

Operating Information

1.

Connect the printer to the RS 232 interface of the control unit (socket „printer“). Note the information for connecting and presetting the printer in the operating manual of the device.

2.

Switch over to the menu for the printer settings, see the menu, the tags and the information about the entries below.

3.

Set the cycle time for the output of data.

4.

Start the printout. Starting and stopping is possible at any time during the process. Operating display : Printer CYCLE: 20 min MODE :start PARAM: __

Symbol / entry

meaning, possible input

CYCLE

Enter cycle time for printout of one line of the protocol in the table. Possible cycle times are in the range of 1 ... 60 minutes.

MODE

Enter operation mode of the printer, select via the „ALTER“ - key. : – stop: no data will be printed – start: printout of data is active

PARAM

Enter 2-digit password „19“ for access to interface parameters.

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Parametrization display for printer: Printer SPEED: 9600 bd DATA : 8 bit STOP : 1 bit next page of parametrization display: Printer PARTY: no 1 : TEMP 2 : STIRR 3 : PH 4 : PO2



Parametrization display with cursor in the lowest line (see print protocol example below) : 7 : 9 : 11: 13:

SUBS1 AIRFL EXT_2 EXT_4

8 : SUBS2 10: EXT_1 12: EXT_3 14:



Symbol / entry

meaning, possible input

SPEED

Enter signal transfer rate within the range of 300..19,200 baud; default is 9,600 baud

DATA

Enter number of data bits: 7 ... 8; default is 8

STOP

Enter number of stop bits: 0 ... 2; default is 1

PARTY

Enter kind of parity bit: „even“, „odd“, „no parity“; default is „no“

1: TEMP. etc.

Selection of a process value for the column 1 ... 14 in the print (possible with software version 4.14 and later versions)

Fig. 5-3: Example for a printer protocol

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5.6.2.3

Host Computers and Connections

The BIOSTAT® B can be connected to a host computer system via the integrated serial interface RS 422. The 4-wire network allows for „Multidrop“ - operation. The communication protocol is compatible with the protocol of the DCU measurement and control system. The data contained are measurement values or setpoints (at fixed order).


Adjustment of interface parameters is possible via protected operation display, entries are accessible via password. The user can define transfer rates within 300 ... 19.200 Baud.

1.

Connect the signal cable to the RS-422 interface on the left side panel of the control unit. Note the documentation of the host computer for the required settings for data transfer.

2.

Switch over the operating terminal to the menu for host communication. Operating display: Host ADR : PARAM:

2 XX

Symbol / entry

meaning, possible input

ADR

Display of BIOSTAT® B - address

PARA

Enter 2-digit password „19“ for access to interface parameters

Operating display „Parameters": Host ADR : 2 SPEED: 9600 bd DATA : 7 bit Operating display page 2, with cursor moved down to the lowest line : Host DATA : 7 bit STOP : 1 bit PARTY: even



Symbol / entry

meaning, possible input

ADR

Enter BIOSTAT® B - address 1 ... 16

SPEED

Enter transfer rate within 300 ... 19,200 baud; default is 9,600 baud

DATA

Enter number of data bits: 7 ... 8; default is 7

STOP

Enter number of stop bits: 0 ... 2; default is 1

PARTY

Enter kind of parity bit: even, odd, no parity; default is „even“

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5.6.3

Utility Functions

Within the „Utility“ - menu you can find and reset, if necessary, some general system settings, such as the „date“ or „real time clock“. The display also shows the implemented software version and any specific configuration, if applied. Operating display, page 1: Utility DATE :17.04.1999 TIME :14:30 h:m VERS : 4.1 Operating display, page 2: Utility CONFG:BA01D 5-4) FAILT:02:00 h:m CONTR:alter



Operating display, page 2, with cursor in the lowest line : Utility FAILT:02:00 h:m CONTR:alter PARAM: ___

5-4)



Symbol / entry

meaning, possible input

DATE

Display or input of date

TIME

Display or input of time (system clock)

VERS

Display of software version

CONFG

Display of configuration identification

FAILT

Display or input of the failtime for power failures. – If a power failure is shorter than the preset „failtime“, the process will be continued with the given settings of all parameters. – If the power failure takes longer, the system behaves like being switched off with the mains switch after return of the power supply.

CONTR

Select contrast of the LCD - display using the „ALTER“ - key.

PARAM

Entry of password for access to the configuration parameters; this is a special password only available for authorized service personnel.

Example for configuration code, see „Utility Functions“ menu of your system for your code. You must refer to this code if you contact B. Braun Biotech International GmbH for questions about your system and if problems arise with your configuration.

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5.6.4

Measurement Ranges

The menu shows the measurement ranges of the process values. Setting of the parameters is possible in a submenu, which is also protected by a password.


Only authorized service personnel is allowed to change the measurement ranges! Operating display, page 1: Meas. Ranges PARAM:___

Page 2 of the Operating Display, with cursor within the upper 4 lines : Meas. Ranges PV :TEMP MIN : 0.0 % MAX : 100.0 %




Symbol / entry

meaning, possible input

PARAM

Enter password „753“ to select the second page of the display.

PV

Display or input of a process value. For selecting another process value use the „ALTER“ - key.

MIN

Display or input of lower limit threshold of a measurement range.

MAX

Display or input of upper limit threshold of a measurement range.

On delivery the following measurement ranges of process values are adjusted as default (you should note that this can be different for the delivered version of the BIOSTAT® B): Process value

TEMP : STIRR: pH : pO2 : ACID : BASE : AFOAM: LEVEL: SU1DC/SU2DC: SUBS1/SUBS2: AIRFL: EXT1....4:

Limits of measurement range lower limit

upper limit

0.0 0 2.00 0.0 0 0 0 0 0 0 0 0

150,0 5-5 1.200 12,00 100,0 500 500 500 10.000 10.000 100 5-6 10.00 100,0

Physical unit

degC rpm pH % ml ml ml ml ml % slpm (lS/min) %

5-5

Max. stirring speed for 2 l - vessel; delivery setting depends on size of the culture vessel and / or is specified according to customized specifications

5 -6

BIOSTAT

B with 5 l culture vessel, may vary for other vessels or customized specifications

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5.6.5

Manual Operation of Digital Outputs, Standard

The menu „Manual Operation - Digital Outputs Std.“ is used for service support. It displays the state of the digital outputs of the system. Inputs in this menu directly trigger the corresponding digital outputs. Menu display : MAN OP DIG 1: a off 3: a off 5: a o

OUT STD 2: a off 4: m 20 6: a off

Symbol / entry

Meaning

1 ... 16

Digital output, channel 1 ... 16

a, etc

Display of mode of output: a : „automatic“: internal function acts on output, i.e. controller o : „operating state“: no internal function acts on output s : „sterilization“: sterilization program acts on output (only available for control systems with sterilization programs, i.e. not valid for BIOSTAT® B, for instance) h : „host“: host computer connected can act on output m : „manual“: manual settings mode of the digital outputs menu enabled; the displayed state will be transmitted

off/on :

Display of state (or manual setting of state in “manual” mode) of: – off: output statically switched off – on: output statically switched on

0 ... 100 :

Display of state (or manual setting of state in “manual” mode) of pulsewidth modulated output : – 0: output switched off – 100: output fully (100%) switched on – 20: output switched on in pulse-with modulated state. In this example the output is switched on at a rating of 20%. The cycle time (pulse - pulse) depends on the controlled system and is in the range of 5 ... 10 s

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5.6.6

Manual Operation of Analog Outputs, Standard

The menu „Manual Operation - Analog Outputs Std.“ is used for service support. It displays the state of the analog outputs of the system. Inputs in this menu directly trigger the corresponding analog outputs. Menu display : MAN OP ANA 1: a o 3: m 3 5: a 10

OUT STD 2: a o 4: a 50 6: a 90

Symbol / entry

Meaning

1 ... 8

analog output, channel 1 ... 8

a, etc

display of mode of output: a : „automatic“: internal function acts on output, i.e. controller o : „operating state“: no internal function acts on output m : „manual“: manual settings mode of the analog outputs menu enabled; the displayed state will be transmitted

0 ... 100

Display of state of output (or manual setting of state in “manual” mode): 0 : output switched off (voltage of 0 V) 100 : output switched on (voltage of 10 V)

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5.7

Alarm Messages of System Functions

When they occur, alarms will be displayed within an extra alarm menu, showing date and time of the alarm. If several alarms occur, they will be stored in an internal buffer, which can hold up to ten alarms. A single alarm message will dissappear after pressing any main function key. For more than one alarm, the next alarm will be displayed afterwards. Example of alarm display : ** ALARM ** Power failure 22.01.91 14:33


another example ** ALARM ** HEATER failtime 22.01.91 14:33

Symbol/entry

meaning, possible input

Heater failure

Display of heater alarm (malfunction of heater)

Motor failure

Display of a motor alarm (malfunction of motor or motor control)

Power failure

Display of general power failure of mains supply

BUFRAM read error

System error; a software reset has been applied or the battery is malfunctioning

EEPROM read error

System error; the parameters specific for the system cannot be processed; not all functions are available (system partly out of order)

How to Answer Alarm Messages : 1.

If the process continuous although an alarm has occured, check which kind of alarm it is and whether the process has been interfered. Disturbances of the process may be indicated at the chart paper of the recorder or the printout of the printer. You may continue the process, if possible or need to interupt it.

2.

If the alarm has stopped the fermentation run, you may try to continue by restarting the fermentor. Check at the display and at the chart paper of the recorder or the printout of the printer at which state of operation the fermentation run has been stopped, which kind of alarm it is and whether the process has been interferred.

3.

At malfunctioning of the related device of fermentor and if the system cannot be restartet: – check all adjustments for the related component – readjust given setpoints, etc., if necessary.




For information about the system parameters adjusted in the factory see section „Factory Settings“ on next page.

4.

If it is impossible for you to restart the fermentor system by this, and in any case one of the system errors „BUFRAM read error“ or „EEPROM read error“ occurs you should contact your B. Braun service representative or directly the B. Braun Biotech International GmbH.

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5.8

Factory Settings

5.8.1

General Information

For the laboratory scale fermentor BIOSTAT® B the integrated measurement and control system offers a defined range of standard configurations. Below you will find a listing of all parameter settings configured in the factory which can be changed by the user according to his specific requirements. In the case of unintended changes of the parameters you can recreate the original state. 5.8.2

Factory Settings for Measurement Ranges of Process Values




Note section “ Measurement Ranges“ further above for an overview of the default settings.




If your fermentor is of customized design and/or has different factory settings, please note the corresponding extra documentation delivered with your equipment. It includes the most actual information on the settings of your BIOSTAT® B.




If your fermentor is of customized design and the corresponding documents are not delivered, please contact B. Braun Biotech International GmbH for this.

5.8.3

Factory Settings for Controller Parameters

5.8.3.1

PID-Parameters und MIN/MAX-Limits

CL-Tag

MIN

MAX

DEADB

XP

TI

TD

RAMP

[%]

[%]

[%]

[%]

[s]

[s]

[%]

TEMP

---

---

0

7

1000

250

---

STIRR

1.7

100

0

200

5

---

---

-100

100

0.5

30

30

0

---

pO2/STIRR

0

100

0,5

150

100

0

---

pO2/AIRFL

0

100

0,5

90

50

0

---

pO2/GASMIX

-100

100

0,5

5

200

0

---

pO2/SUBS1

-100

100

---

120

60

0

---

0

100

---

---

---

---

---

-100

100

---

---

---

---

---

0

100

---

---

---

---

---

pH

SUBS1 / 2 GASMIX AIRFL




If your fermentor is of customized design and/or has different factory settings, please note the corresponding extra documentation delivered with your equipment. It includes the most actual information on the settings of your BIOSTAT® B.




If the corresponding documents are not delivered with the unit, please contact B. Braun Biotech International GmbH for this.

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5.8.4

Example: pO2-Controller with Airflow - Massflow Controller and O2 - Enrichment




After switching on the pO2 - controller first operates the AIRFL - controller. At this time the GASMIX - controller receives the „MIN“ setting assigned in the pO2 - controller.




If the setpoint setting of the AIRFL - controller reaches its maximum, the output of the pO2 controller switches over to the setpoint input of the GASMIX - controller, see fig. on the next page. The AIRFL - controller receives the „MAX“ setting defined in the pO2 - controller. For control of the pO2 the GASMIX - controller operates a 3/2-way valve for supply of air and O2. Operating display: pO2 SETP MODE CASC

: : : :

88.5 % 83.4 % auto AIRFL

GASMX

Symbol / entry

meaning, possible input

pO2

Display of actual value des Istwertes

SETP

Display and input of the controller´s setpoint

MODE

Display and input of the controller´s operating mode: – auto: automatic operation with the given setpoint; – off: controller is switched off; outputs are in operating state

CASC

Display and selection of servo controller 1 and 2: AIRFL : The pO2 - controller operates the setpoint of the Airflow - controller GASM : The pO2 - controller operates the setpoint of the Gasmix-controller

PARAM 5.8.4.1

Access to the controller parameters via 2-digit password „19“

Parametrization Parametrization display page 1 : pO2 : 88,4 % DEADB: 0.5 % HTIME: 5 min MIN : 0 %

ñ

GASMX

Parametrization display page 2 : pO2 MAX XP TI

: 88.4 % : 100 % : 100 s : 100 s

ñ

Symbol / entry

meaning, possible input

DEADB

Display and input of the Totzone („Dead band“)

HTIME

Display and input of delay time for switching from cascade 1 to 2

AIRFL, etc.

Selction of the servo controller using the ALTER key

AIRFL : Display and input of the parameters for the Airflow - controller GASM : Display and input of the Parameter for the Gasmix - controller

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Operating Manual BIOSTAT B

5 Control Unit

5.8.4.2

Schematic Drawing, Set-up of „Oxygen Enrichment System“

Fig. 5-4:

Set-up of „Oxygen Enrichment System“

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5.8.5


Schematic Drawing „Pump Logic of DFC - Software“ The schematic drawing below shows the different modes of pump control: pH

The pH - controller operates the ACID - pump; – switching over is possible for control by the SUBS1/2 - controllers

pH

The pH - controller operates the BASE - pump; – switching over is possible for control by the SUBS1/2 - controllers

FOAM

The antifoam controller operates the AFOAM - pump; – switching over is possible for control by the SUBS1/2 - controllers

LEVEL

The level - controller operates the LEVEL – pump; – the level controller can be switched over to the modes „Harvest“ or „FEED“ for operation of the LEVEL - pump; – switching over is possible for control by the SUBS1/2 - controllers

SUBS1/2

The substrate - controllers operate the external substrate pump; – the substrate controller can be switched over for cascade operation by the pO2 - controller (CASC) and profil mode (PROF): – switching over is possible for operation of the ACID, BASE, AFOAM or LEVEL - pump

Fig. 5-5: Pump switching logic of DFC Software

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Operating Manual BIOSTAT B

5 Control Unit

5.8.6


5.8.7


Arrangement Plan Control Unit of BIOSTAT® B See drawing(s) on the next page(s). If the arrangement plan(s) do(es) not match with the BIOSTAT® B delivered to you and if the customized documentation is not delivered to you with the equipment or by extra mail, please contact B. Braun Biotech International GmbH for an update of the documentation. Assignment Plan for Interfaces of the Control Unit BIOSTAT® B See drawing(s) on the next page(s). If the assignment plans do not match with the BIOSTAT® B delivered to you and if the customized documentation is not delivered to you with the equipment or by extra mail, please contact B. Braun Biotech International GmbH for an update of the documentation.

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Technical Data

6

TECHNICAL DATA

6.1

Dimensions


Basic unit




Culture vessel (W x H x D) :

type B2, 305 x 580 x 270 mm (with stand and motor); type B5, 400 x 685 x 330 mm (with stand and motor); type B10, 420 x 828 mm (W x H; with stand and motor)




Weight

:

control unit approx. 30 kg; type B2: approx. 16 kg, completely equipped; type B5: approx. 25 kg, completely equipped; type B10: depending on equipment

6.2

:

365 x 536 x 450 mm (W x H x D)

Installations


Mains connection

:

230 V / 50 ... 60 Hz 115 V / 50 ... 60 Hz




Power consumption

:

max. 900 W




Cooling water

:

2 barg (about 29 psig), controlled, connector ID 12 mm; cooling water flow max. 5 l/min




Cooling water outlet

:

Connector ID 8 mm




Air inlet

:

0.8 barg, controlled, connector ID 8 mm, air flow max. 10 l/min

6.3

Control unit


Thermostate system

:

open thermostate system with circulation pump; heater of 600 or 800 W, with solenoid valve; temperature range 8 degC above cooling water temperature in the range up to 60 degC




Drive system

:

electronic motor 180 W, speed range 50 ... 1,200 rpm (need be limitted to 50 ... 600 rpm for 10 l culture vessels by setpoint adjustment)




Aeration system

:

autoclavable membrane filter, 0,2 µm, for sterile aeration and exhaust; rotameter, manual control valve, air flow max. 10 l/min optional airflow control equipment




Corrective solution pump

:

4 built-in peristaltic pumps for acid, alkali, antifoam, harvest/feed, substrate 1, substrate 2; flow rate max. 8 ml/min for tubings size 1.6 x 1.6 mm; max. 30 ml/min for tubings size 3.2 x 1.6 mm




Storage bottle

:

3 glass bottles 250 ml for B 2 and B2-airlift; 3 glass bottles 500 ml for B 5, B5-airlift and B 10

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Technical Data

6.4

Culture Vessels

6.4.1

Culture Vessel Type B 2




Cat.-no.

:

884 036/9




Set up

:

jacketed glass vessel with stainless steel-top plate and baffle insert; total volume 3 l, working volume 2 l




Materials

:

glass vessel made of borosilicate glass - steel parts in touch with medium made of stainless steel 1.4571 (corresponding with US-standard 316TI), - other steel parts steel 1.4301 (US-standard 304TI) - seals made of EPDM (ethylen-propylen rubber)




Stirring system

:

-




Ports

:

4 side ports in the head space of the glass wall 2 top plate ports Ø 19 mm; 9 top plate ports Ø 6 mm; 4 top plate hose connectors Ø 4 mm; 2 top plate ports Ø 12 mm (Pg 13,5)




Autoclave size (min. required) :

6.4.1.1

rotary shaft with viton lip seals (until January 1993); actual design: rotary shaft with mechanical seal; stirrer speed range 50 ... 1200 rpm; two 6-bladed-disk impellers 6-1)

;

Height 550 mm, diameter 360 mm

Standard Equipment

Cat.-no.

Device

3821963/8 baffle insert

Description, specification vessel insert with 4 baffles

3821786/4 aeration pipe with sparger ring 881 007/9

plastic filter for air inlet and exhaust membrane filter, by Millipore

3824042/4 exhaust cooler B2 sampling system

harvesting pipe with top-plate adapter

3319088/7 temperature sensor Pt 100

type 200-4

3318258/2 pH-electrode with filling of pressurized pasty electrolyte

type Pa/12, length 200 mm

3409978/6 pO2-electrode

type 12/220

0884446/1 antifoam probe

immersion depth 50 mm

0884448/8 level probe

immersion depth 122 mm

3821785/6 flat bladed stirrers with 6 blades

delivery set of 2 pcs.

manual sampler 0882360/0 storage bottles, 250 ml

6-1)

delivery set of 3 pcs.

Glass vessels are optionally available without side-entry ports, further information on request.

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Technical Data

6.4.1.2

Optional Equipment for Culture Vessel B2

Cat.-no.

Device

Description, specification

884 432/1

thermometer kit for vessel B2

for visual temperature monitoring; with pocket

3823907/8 paddle stirrer for vessel B2

delivery of 2 paddle stirrers with option „silicone membrane insert“

3833697/9 universal hose connector

adapter for 1 supply to the culture vessel

884 431/3

adapter for 4 supplies to the culture vessel

quadruple hose connector B

3833717/7 reduction connector 19/6

adapter for mounting of devices with Øa = 6 mm in top-plate ports 19 mm

884 463/1

inoculation kit B septum with blind closure and septum membrane

for inoculation or dosing of substrate via syringe; membrane is self-sealing and can be punctured several times

884 459/3

flexible holder for exhaust cooler

with this adapter the exhaust cooler can be bent over for autoclave sterilization, this reduces the height required for the autoclave the

884 434/8

Bypass sampler B set for assembly of a bypass

-

884 438/0

harvest filter B2 for removal of samples at microcarrier cultures

- rising pipe with teflon filter tube, - pore width 25 µm

884 466/6

kit „silicone membrane insert B5 for bubble-free gas supply“

-

silicone membrane, L = 5,2 m, Ø = 3 x 3,7 mm membrane filter for gas inlet and outlet, 2 pcs. pressure controller 2 paddle stirrers

880 830/9

spinfilter B2, 20 µm

-

mounted to stirrer shaft 4-layer mesh made of stainless steel 1.4404 pore width 20 µm harvesting pipe 880826/0 required

880 831/7

spinfilter B2, 40 µm

- as above, but with pore width 40 µm

880832/5

spinfilter B2, 75 µm

- as above, but with pore width 75 µm

880 826/0

harvesting pipe SF B2

for sample removal via spinfilter

884 436/4

perfusion module B2

perfusion system for continuous fermentation, with microporous membrane

884041/5

guiding tube for B2 culture vessel

recommended for assembly with perfusion module and spinfilter, if no silicone membrane insert is used

6.4.1.3 Cat.-no.

membrane holder with septum membrane 2-channel top-plate adapter 19 mm 2 m silicone tubing 1.6 x 1.6 mm 0,2 m PTFE-Schlauch 3,5 x 5,5 mm

Spare Parts for Culture Vessel B2 Device

Description, specification

3920427/8 Spare glass vessel B2

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Technical Data

6.4.2

Culture Vessel Type B 5




Cat.-no.

:

884 037/7




Set up

:

jacketed-glass vessel with stainless steel-top plate and baffle insert, total volume 6,6 l, working volume 5 l




Materials

:

glass vessel made of borosilicate glass - steel parts in touch with medium made of stainless steel 1.4571 (corresponding with US-standard 316TI), - other steel parts steel 1.4301 (US-standard 304TI) - seals made of EPDM (ethylen-propylen rubber)




Stirring system

:

-




Ports

:

4 side ports in the head space of the glass wall 2 top plate ports Ø 19 mm; 9 top plate ports Ø 6 mm; 4 top plate hose connectors Ø 4 mm; 2 top plate ports Ø 12 mm (Pg 13,5)




Autoclave size (min. required) :

6.4.2.1

rotary shaft with viton lip seals (until January 1993); actual design: rotary shaft with mechanical seal; stirrer speed range 50 ... 1200 rpm; three 6-bladed-disk impellers 6-2)

;

Height 700 mm, diameter 400 mm

Standard Equipment

Cat.-no.

Device

Description, specification

38219670

baffle insert

38218569

aeration pipe with sparger ring

881 007/9

plastic filter for air inlet and exhaust membrane filter, by Millipore

3824116/1 exhaust cooler B5 sampling system 33190895

temperature sensor Pt 100

type 300-4

3318257/4 pH-electrode with filling of pressurized pasty electrolyte

type Pa/12, length 325 mm

3409979/4 pO2-electrode

type 12/320

0884446/1 antifoam probe

immersion depth 50 mm

0884448/8 level probe

immersion depth 122 mm

38218550

delivery set of 3 pcs.

flat bladed stirrers with 6 blades manual sampler

0882361/8 storage bottles, 500 ml

6-2)

delivery set of 3 pcs.

Glass vessels are optionally available without side-entry ports, further information on request.

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6

Operating Manual BIOSTAT B

Technical Data

6.4.2.2

Optional Equipment for Culture Vessel B5

Cat.-no.

Device

Description, specification

884 453/4

thermometer kit for vessel B5

for visual temperature monitoring; with pocket

3823954/0 paddle stirrer for vessel B5

delivery of 2 paddle stirrers with option „silicone membrane insert“

3833697/9 universal hose connector

adapter for 1 supply to the culture vessel

884 431/3

adapter for 4 supplies to the culture vessel

quadruple hose connector B

3833717/7 reduction connector 19/6

adapter for mounting of devices with Øa = 6 mm in top-plate ports 19 mm

884 463/1

inoculation kit B; septum with blind closure and septum membrane

for inoculation or dosing of substrate via syringe; membrane is self-sealing and can be punctured several times

884 459/3

flexible holder for exhaust cooler

with this adapter the exhaust cooler can be bent over for autoclave sterilization, this reduces the height required for the autoclave

884 434/8

Bypass sampler B set for assembly of a bypass

-

884457/7

harvest filter B5 for removal of samples at microcarrier cultures

- rising pipe with teflon filter tube, - pore width 25 µm

884 467/4

kit „silicone membrane insert B5 for bubble-free gas supply“

-

silicone membrane, L = 10,4 m, Ø = 3 x 3,7 mm membrane filter gas inlet and outlet, 2 pcs. pressure controller 2 paddle stirrers

880 833/3

spinfilter B5, 20 µm

-

mounted to stirrer shaft 4-layer mesh made of stainless steel 1.4404 pore width 20 µm harvesting pipe 880826/0 required

880 834/1

spinfilter B5, 40 µm

- as above, but with pore width 40 µm

880 835/0

spinfilter B5, 75 µm

- as above, but with pore width 75 µm

880 827/9

harvesting pipe SF B5

for sample removal via spinfilter

884439/9

perfusion module B5

perfusion system for continuous fermentation, with microporous membrane

884 066/0

guiding tube for B5 culture vessel recommended for assembly with perfusion module and spinfilter, if no silicone membrane insert is used

6.4.2.3 Cat.-no.

membrane holder with septum membrane 2-channel top-plate adapter 19 mm 2 m silicone tubing 1.6 x 1.6 mm 0,2 m PTFE-Schlauch 3,5 x 5,5 mm

Spare Parts for Culture Vessel B5 Device

Description, specification

3920428/6 Spare glass vessel B5

BAE_B Rev. 2.31 - 1099 SW 4.1

6-5

6

Operating Manual BIOSTAT B

Technical Data

6.4.3

Culture Vessel Type B 10




Cat.-no.

:

884 038/5




Set up

:

jacketed-glass vessel with stainless steel-top plate and baffle insert, total volume 13 l, working volume 5-10 l




Materials

:

glass vessel made of borosilicate glass - steel parts in touch with medium made of stainless steel 1.4571 (corresponding with US-standard 316TI), - other steel parts steel 1.4301 (US-standard 304TI) - seals made of EPDM (ethylen-propylen rubber)




Stirring system

:

- rotary shaft with mechanical seal; - stirrer speed range 50 ... 800 rpm; - three 6-bladed-disk impellers




Ports

:

4 top plate ports Ø 19 mm; 9 top plate ports Ø 6 mm; 4 top plate hose connectors Ø 4 mm; 2 top plate ports Ø 12 mm (Pg 13,5)




Autoclave size (min. required) :

6.4.3.1

Height 820 mm, diameter 420 mm

Standard Equipment:

Cat.-no.

Device

38241072

baffles

38241080

aeration pipe with baffle

sparger ring at lower end

881 007/9

plastic filter for Zuluft

membrane filter, by Millipore

3922830/4 membrane-filter tube for Abluft

Description, specification

filter tube Emflon II,, by Pall

3824116/1 exhaust cooler B5 sampling system 00001769

temperature sensor Pt 100

3318257/4 pH-electrode with filling of pressurized pasty electrolyte

type Pa/12, length 325 mm

3409979/4 pO2-electrode

type 12/320

884446/1

antifoam probe

immersion depth 50 mm

884448/8

level probe

immersion depth 122 mm

38214881

flat bladed stirrers with 6 blades

delivery set of 3 pcs.

manual sampler 0882361/8 storage bottles, 500 ml

BAE_B Rev. 2.31 - 1099 SW 4.1

delivery set of 3 pcs.

6-6

6

Operating Manual BIOSTAT B

Technical Data

6.4.3.2

Optional Equipment for Culture Vessel B10

Cat.-no.

Device

Description, specification

884 453/4

thermometer kit

for visual temperature monitoring; with pocket

884 054/7

paddle stirrers

delivery of 2 paddle stirrers with option „silicone membrane insert“

3833697/9 universal hose connector

adapter for 1 supply to the culture vessel

884 431/3

adapter for 4 supplies to the culture vessel

quadruple hose connector B

3833717/7 reduction connector 19/6

adapter for mounting of devices with Øa = 6 mm in top-plate ports 19 mm

884 463/1

inoculation kit B septum with blind closure and septum membrane

for inoculation or dosing of substrate via syringe; membrane is self-sealing and can be punctured several times

884 459/3

flexible holder for exhaust cooler

with this adapter the exhaust cooler can be bent over for autoclave sterilization, this reduces the height required for the autoclave the

884 434/8

Bypass sampler B; set for assembly of a bypass

-

884 060/1

inoculation kit B10

septum port assembly for inoculation connector cat.no. 884 061/0

884 061/0

1-inlet inoculation connector for inoculation kit B10

- hollow needle Ø 6 x 0,5 mm made of stainless steel 1.4435, with sterile sleeve

884 050/4

harvest filter B10 for sample removal at microcarrier cultures

- rising pipe with filter tube, pore width 25 µm

884 048/2

kit „silicone membrane insert B10 - silicone membrane, L = 16 m, Ø = 3 x 3,7 mm for bubble-free gas supply“ - membrane filter for gas inlet and outlet - pressure controller - 2 paddle stirrers

884 055/5

spinfilter B10, 20 µm

-

884 056/3

spinfilter B10, 40 µm

as above, but with pore width 40 µm

884057/1

spinfilter B 10, 75 µm

as above, but with pore width 75 µm

884 059/8

harvesting pipe SF B10

for sample removal via spinfilter

884 049/0

perfusion module B10

perfusion system for continuous fermentation, with microporous membrane

884058/0

guiding tube for culture vessel B10

recommended for assembly with perfusion module and spinfilter, if no silicone membrane insert is used

BAE_B Rev. 2.31 - 1099 SW 4.1

membrane holder with septum membrane 2-channel top-plate adapter 19 mm 2 m silicone tubing 1.6 x 1.6 mm 0,2 m PTFE-Schlauch 3,5 x 5,5 mm

mounted to stirrer shaft 4-layer mesh made of stainless steel 1.4404 pore width 20 µm harvesting pipe 880826/0 required

6-7

6

Operating Manual BIOSTAT B

Technical Data

6.4.3.3

Spare Parts for Culture Vessel B10

Cat.-no.

Device

Description, specification

3920418/9 Spare glass vessel B10

6.4.4

Culture Vessel B 10, Special Version for Small Working Volumes




Cat.-no.

:

884 039/3




Setup

:

jacketed-glass vessel with stainless steel-top plate; total volume 13 l, working volume 1,5 - 10 l




Equipment:

Cat.-no.

Device

Description, specification

00001769

temperature sensor Pt 100

type 400-4

3409989/1 pH-electrode with filling of pressurized pasty electrolyte

type Pa/12, length 425 mm

pO2-electrode

type 12/420

level probe

immersion depth 300 mm

BAE_B Rev. 2.31 - 1099 SW 4.1

6-8

6

Operating Manual BIOSTAT B

Technical Data

6.4.5

Culture Vessel B 2 - Airlift




Cat.-no.

:

884 044/0




Setup

:

jacketed-glass vessel with concave bottom - H:W (w.v.) = 4 : 1, H:W (total) = 5,4 : 1 - total volume 3,5 l, working volume 2 l - loop reactor with guiding tube instead of stirrer drive




Materials

:

glass vessel made of borosilicate glass - steel parts in touch with medium made of stainless steel 1.4571 (corresponding with US-standard 316TI), - other steel parts steel 1.4301 (corresponding with USstandard 304TI) - seals made of EPDM (ethylen-propylen rubber)




Ports

:

5 top plate ports Ø 19 mm; 6 top plate ports Ø 6 mm; 4 top plate hose connectors Ø 4 mm




Autoclave size (min. required) :

6.4.5.1

Height 700 mm, diameter 350 mm

Standard Equipment

Cat.-no.

Device

Description, specification

38219395

guiding tube with aeration pipe

sparger ring at lower end

881 007/9

plastic filter for air inlet and exhaust

membrane filter, by Millipore

3824042/4 exhaust cooler B2 sampling system 33190895

temperature sensor Pt 100

type 300-4

3318257/4 pH-electrode with filling of pressurized pasty electrolyte

type Pa/12, length 220 mm

3409979/4 pO2-electrode

type 12/220

0884446/1 antifoam probe

immersion depth 50 mm

0884448/8 level probe

immersion depth 50 mm

manual sampler 0882360/0 storage bottles, 250 ml

BAE_B Rev. 2.31 - 1099 SW 4.1

delivery set of 3 pcs.

6-9

6

Operating Manual BIOSTAT B

Technical Data

6.4.5.2

Optional Equipment for Culture Vessel B2 - Airlift

Cat.-no.

Device

Description, specification

884 432/1

thermometer kit for vessel B2

for visual temperature monitoring; with pocket

3833697/9 universal hose connector

adapter for 1 supply to the culture vessel

884 431/3

adapter for 4 supplies to the culture vessel

quadruple hose connector B

3833717/7 reduction connector 19/6

adapter for mounting of devices with Øa = 6 mm in top-plate ports 19 mm

884 463/1

inoculation kit B/MD, septum with for inoculation or dosing of substrate via syringe; blind closure and septum memmembrane is self-sealing and can be punctured sevbrane eral times

884 459/3

flexible holder for exhaust cooler

with this adapter the exhaust cooler can be bent over for autoclave sterilization, this reduces the height required for the autoclave the

884 434/8

Bypass sampler B; set for assembly of a bypass

-

6.4.5.3 Cat.-no.

membrane holder with septum membrane 2-channel top-plate adapter 19 mm 2 m silicone tubing 1.6 x 1.6 mm 0,2 m PTFE-Schlauch 3,5 x 5,5 mm

Spare Parts for Culture Vessel B2 - Airlift Device

Description, specification

3920429/4 Spare vessel B2 - airlift

BAE_B Rev. 2.31 - 1099 SW 4.1

6 - 10

6

Operating Manual BIOSTAT B

Technical Data

6.4.6

Culture Vessel B 5 - Airlift




Cat.-no.

:

884 045/8




Setup

:

same as culture vessel B2 - airlift, except - H:W (w.v.) = 3,7 : 1, H:W (total) = 5,0 : 1 - total volume 9 l, working volume 5 l




Materials

:

same as culture vessel B2 - airlift




Ports

:

3 top plate ports Ø 19 mm; 10 top plate ports Ø 6 mm; 4 top plate hose connectors Ø 4 mm; 2 top plate ports Ø 12 mm (Pg 13,5)




Autoclave size (min. required) :

6.4.6.1

Height 900 mm, diameter 420 mm

Standard Equipment:

Cat.-no.

Device

Description, specification

n.n.

guiding tube

acc. to drwg. B318.27.04.00.104

n.n.

aeration pipe, sparger ring at lower end

acc. to drwg. B318.27.02.00.104

881 007/9

plastic filter for air inlet and exhaust

membrane filter, by Millipore

3824116/1 exhaust cooler B5 sampling system n.n.

temperature sensor Pt 100

on request

3318257/4 pH-electrode with filling of pressurized pasty electrolyte

type Pa/12, length 325 mm

3409979/4 pO2-electrode

type 12/320

0884446/1 antifoam probe

immersion depth 50 mm

0884448/8 level probe

immersion depth 122 mm

manual sampler 0882361/8 storage bottles, 500 ml

BAE_B Rev. 2.31 - 1099 SW 4.1

delivery set of 3 pcs.

6 - 11

6

Operating Manual BIOSTAT B

Technical Data

6.4.6.2

Optional Equipment for Culture Vessel B5 - Airlift

Cat.-no.

Device

Description, specification

884 453/4

thermometer kit B5

for visual temperature monitoring; for mounting in thermometer pocket

3833697/9 universal hose connector

adapter for 1 supply to the culture vessel

884 431/3

adapter for 4 supplies to the culture vessel

quadruple hose connector B

3833717/7 reduction connector 19/6

adapter for mounting of devices with Øa = 6 mm in top-plate ports 19 mm

884 463/1

inoculation kit B / MD septum with blind closure and septum membrane

for inoculation or dosing of substrate via syringe; membrane is self-sealing and can be punctured several times

884 459/3

flexible holder for exhaust cooler

with this adapter the exhaust cooler can be bent over for autoclave sterilization, this reduces the height required for the autoclave the

884 434/8

Bypass sampler B; set for assembly of a bypass

-

6.4.6.3

membrane holder with membrane 2-channel top-plate adapter 19 mm 2 m silicone tubing 1.6 x 1.6 mm 0,2 m PTFE-Schlauch 3,5 x 5,5 mm

Spare Parts for Culture Vessel B5 - Airlift

Cat.-no.

Device

Description, specification

n.n.

spare glass vessel B5 - airlift

accord. to drwg. B318.27.00.06.103

BAE_B Rev. 2.31 - 1099 SW 4.1

6 - 12

6

Operating Manual BIOSTAT B

Technical Data

6.5

Additional Equipment

Cat.-no.

Device

880 924/0

STT-quick connector, female con- - for sterile tubing connections nector of stainless steel for tubings - delivery with blind closure Ø = 3.2 x 1.6 mm - connection at culture vessel

880 920/8

STT-quick connector, male connector for tubings 3.2 x 1.6 mm

counterpiece to female connector 880 924/0 - delivery with cap - connection der externen Zuleitung

880 941/0

STT-quick connector, female connector for tubings Ø = 1.6 x 1.6

like STT - female connector 880 924/0

880 940/2

STT-quick connector, male connector for tubings Ø = 1.6 x 1.6

counterpiece to female connector 880 941/0 like STT-male connector 880 920/8

882364/2

Storage and harvesting bottle 10 ltr., autoclaveable

-

882365/0

Storage and harvesting bottle 20 ltr., autoclaveable

large bottle made of polypropylen, 20 l, equipment as shown above, delivered with 0,7 m of silicone tubing 3.2 x 1.6 mm

882366/9

Storage and harvesting bottle 50 ltr., autoclaveable

large bottle made of polypropylen, 50 l, equipment as shown above, delivered with 2 m of silicone tubing 3.2 x 1.6 mm,

6.6

Description, specifications

large bottle made of polypropylen, 10 l stainless steel headpiece with 3 inlets silicone tubing 3.2 x 1.6 mm, 40 cm length top-plate screw with washer deaeration filter

Consumables

Cat.-no.

Device

Description, specifications

618201/1

silicone tubing 1.6 x 1.6 mm

minimum order 10 m

3997115/5 silicone tubing 3.2 x 1.6 mm

minimum order 10 m

3997078/7 PTFE-tube 880 926/7

slotted membrane for connection of STT - quick connector

for STT-female connector 880924/0 or 880941/0; kit with 20 pcs.

3925071/7 pH calibration buffer pH 4

bottle 250 ml

3925072/5 pH calibration buffer pH 7

bottle 250 ml

3925073/3 pH calibration buffer pH 9

bottle of 250 ml

3409947/6 membrane kit for pO2-electrode

for electrodes with Ø = 12 mm

882157/7

bottle of 50 ml

elektrolyte for pO2-electrode

3922480/5 filter

for storage bottle and manual sampler

3880485/9 septum membrane (washer)

for Bypass- sampler, set of 10 pcs.

882 165/8

Pepsin-HCl - cleaning agent

bottle of 250 ml

882 166/6

Thio-Urea - cleaning agent

bottle of 250 ml

BAE_B Rev. 2.31 - 1099 SW 4.1

6 - 13

6

Operating Manual BIOSTAT B

Technical Data

6.7

Control Unit


System set-up

:

highly integrated microprocessor-board (DFC-2) with integrated measurement amplifiers and actuator controls; measurement amplifiers for temperature, pH, pO2, foam and level; microprocessor-system with 80C188 processor, offering - 128 kByte RAM, 128 kByte EPROM, - 8 analogue inputs, 8 analogue outputs, - 8 digital inputs and 16 digital outputs; automatic restart after power down




Operating panel

:

illuminated LCD-Display with 4 lines x 20 characters; Film keyboard with 20 keys




Temperature control

:

sensor Pt 100, measurement range 0 ... 100 degC digital PID-controller with split range outputs for heating and cooling




Stirrer-control

:

RPM-measurement with Hall sensors, - measurement range 0 ... 1,200 rpm digital PID-controller for external motor controller




pH-control

:

Ingold pH-electrode with pasty electrolyte (Ø 12 mm) digital sensor calibration measurement range 2 ... 12 pH digital PID-controller with split range outputs for acid and alkali dosing counter for acid-/alkaline agent - pump




pO2-control

:

Ingold pO2-Electrode (Ø 12 mm); digital sensor calibration; measurement range 0 ... 100 % pO2 (with reference to oxygen saturation of the measurement solution); digital PID-cascade controller for stirrer speed sequential controller or for control of a Gas Mix unit (optional)




Antifoam-Control

:

conductive foam sensor; digital on/off controller for antifoam agent pump; dosing counter for antifoam agent pump




Level control

:

conductive level sensor; digital liwith controller for harvest or substrate pump dosing counter for harvest/substrate pump




Substrate pump control

:

max. 2 pumps via pulse width modulated outputs; adjustable delivery rate 0 ... 100%; dosing counters for feed pumps




Recorder connection

:

6 analogue outputs 0 ... 10 V; assignment of parameters to channel configurable




Protocol printer connection :

via serial interface RS 232 C; printing intervals and process variables for protocol configurable




Host connection

via serial interface RS 422; protocol with measured values, set points and controller mode

BAE_B Rev. 2.31 - 1099 SW 4.1

:

6 - 14

7

Operating Manual BIOSTAT B

Optional Equipment and Accessories

7

OPTIONAL EQUIPMENT AND ACCESSORIES


Detailed information about optional equipment and accessories will be included herein by time oder will be sent to you together with the equipment or by extra mail.




If your fermentor system BIOSTAT® B includes optional equipment or is of special design, which is not described in the above manual or by the documentation of this section or as extra delivered, please contact B. Braun Biotech International GmbH for such documentation:




B. Braun Biotech International GmbH Schwarzenberger Weg 73-79 D - 34212 Melsungen, Fed. Republic of Germany phone +49 56 61 - 71 34 00, fax +49 56 61 - 71 37 02 e-mail: [email protected] WebSite http://www.bbraunbiotech.com

BAE_B Rev. 2.31 - 1099 SW 4.1

7-1

8

Operating Manual BIOSTAT B

Supplement

8

SUPPLEMENT

8.1

Conversion Table for Degrees of Hardness of Water Note: In this table the german decimal comma (",") is used instead of the english decimal point (".") alkaline alkaline earth ions earth ions

german degrees

CaCO3

engl. degrees

french. degrees

[mmol/l]

[mval/l]

[°d]

[ppm]

[°e]

[°f]

1 mmol/l alkaline earth ions

1,00

2,00

5,50

100,00

7,02

10,00

1 mval/l alkaline earth ions

0,50

1,00

2,80

50,00

3,51

5

1 german degree [°d]

0,18

0,357

1,00

17,80

1,25

1,78

1 ppm CaCO3

0,01

0,020

0,056

1,00

0,0702

0,10

1 engl. degree [°e]

0,14

0,285

0,798

14,30

1,00

1,43

1 french degr. [°f]

0,10

0,200

0,560

10,00

0,702

1,00

8.2

Characteristic Values for Gases Gas

:

Density

Carbon dioxide CO2

:

1,977 kg/m

3

Air

:

1,293 kg/m

3

Methane

:

0,717 kg/m

3

Oxygen

:

1,429 kg/m

3

Nitrogen

:

1,251 kg/m

3

BAE_B Rev. 2.31 - 1099 SW 4.1

8-1

8

Operating Manual BIOSTAT B

Supplement

8.3

Calculation Methods for Flowrates of the Rotameters

8.3.1

Sizing of Flowmeters / Rotameters

Rotameters of the Gas Mixing System or flowmeters are usually calibrated and graduated according to standard conditions. These calibration conditions are marked on every glass tube or on the support connector of any rotameter. For instance, such calibration conditions may be


Calibration gas

:

air




Temperature

:

20 °C = 293 K




Pressure

:

1 bar overpressure

->

Please check which values are marked on your rotameters / flowmeters and must be considered for further evaluations.

As far as your operation conditions (type of gas, density, temperature, pressure, etc.) deviate from the standard conditions and if you need the exact flowrates for the interpretation of the fermentation process, you may calculate the reference flowrates using the procedures or the tables / nomographes shown below. Additional information about calculation of flowrates of a gas under special conditions of temperature, pressure and specific density (specific heat) can be found in the corresponding literature or is available from manufacturers of rotameters / flowmeters. You may also use these formulas, tables or nomographes to evaluate which design of the Gas Mixing System will meet your specific requirements. For selection of an appropriate Gas Mixing System it is important, that its design allows the maximum flowrates of the individual gas which are required for the projected fermentation process.


If the rotameters are inadequately sized delivering to small flowrates, the automatic control may not function properly.




Especially, when the pO2-Control with the gas supply does not operate accurately, this may be due to wrong sized rotameters.

8.3.2

Calculation Method

The corrective calculation for the flowrates includes the following formula (1) - (3) : (1)

q1

=

q2 * F

q1

=

air-equivalent flowrate of the gas (flowrate read on the flowmeter

q2

=

actual flowrate of the gas

F

=

conversion factor

BAE_B Rev. 2.31 - 1099 SW 4.1

8-2

8

Operating Manual BIOSTAT B

Supplement

According to Brooks, „Sizing calculations“, the conversion factor F can be calculated by equation (2) :

F= ρ1 ρ2 T1 T2 p1 p2

= = = = = =

ρ2 · T2 · p1 ρ1· T1· p2

density of the calibration gas; e.g. for air the density is ρ1 = 1,293 kg/m density of the gas to be controlled 8-1) temperature in [Kelvin] at calibration conditions temperature at measurement conditions, in [Kelvin] pressure at calibration conditions, in [bar] pressure at measurement conditions, in [bar]

3

This leads to equation (3):

q2 =

q1 = F

q1 ρ2 · T2 · p1 ρ1· T1· p2

Example for calculating: Evaluated gas

:

for calibration : air ; 3 density (20 °C) ρ1 = 1,293 kg/m gas for measurement CO2; 3 density (20 °C) ρ2 = 1,977 kg/m

Standard flow

:

q1 = 10 l/min

Pressure

:

for calibration : p1 = 1 barabs operation pressure : p2 = 1,5 barabs

Temperatur e

:

for calibration : 20 °C (= 293 K) operation temperature 25 °C (= 298 K)

These values give the following result for the actual flow of CO2 at operating conditions :

q2(CO2) =

10 l / min = F

10 l / min 1977 , · 298 · 1 1293 , · 293 · 15 ,

q2 (CO2) = 9,8 l/min

8.3.3

8-1)

Manufacturer's Examples for Sizing Calculations




The following tables and nomographes give examples of manufacturer's of metering tubes and rotameters for calculation of actual flowrates at given operating conditions.




If you are using other brands or types of flowmeters / rotameters, you may find corresponding information in the documentation delivered with your specific equipment or you may contact the manufacturer of these parts for further support.

Densities of usual gases applied in fermentation processes can be found in the above table "Characteristic Values for Gases" as well as in corresp. literature

BAE_B Rev. 2.31 - 1099 SW 4.1

8-3

Operating Manual BIOSTAT B

8 Supplement

BAE_B Rev. 2.31 - 1099 SW 4.1

8-4

Operating Manual BIOSTAT B

8 Supplement

BAE_B Rev. 2.31 - 1099 SW 4.1

8-5

8

Operating Manual BIOSTAT B

Supplement

8.4

Additional Drawings and Flow Charts

8.4.1

P & I - Diagram of the BIOSTAT® B




8.4.2


See copy attached on next page. This is an example of the default design of the BIOSTAT® B at the time this document was printed.

Drawings and Flow-Charts of Fermentor Options or Customized Fermentors

Drawings will be completed with delivery of the manual, especially when drawings are available of optionally designed fermentor systems BIOSTAT® B.

BAE_B Rev. 2.31 - 1099 SW 4.1

8-6

Operating Manual BIOSTAT B

8 Supplement

Declaration of Conformity with EC-Directives, Standards and Technical Specifications

By the copies of the „Declaration of Conformity“ enclosed in this Operating Manual or delivered extra with the device the B. Braun Biotech International GmbH confirms, that the fermentor systems BIOSTAT® B fulfill the requirements of all mentioned EC-directives, harmonized and national standards and technical specifications. The signatures of the English forms substitutionally confirm the validity of the declaration for all other language forms.

BAE_B Rev. 2.31 - 1099 SW 4.1

8-7