ez-control for Autoclavable Bioreactors 1 20 L HARDWARE MANUAL

ez-control for Autoclavable Bioreactors 1 20 L

SYMBOLS SYMBOLS The following symbols are used on the equipment and in this manual: Warning: refer to this manual. Caution: electrical shock hazard. Important note: read the instructions carefully. Risk of physical injury. Additional Information. Lifting instructions. Applikon Biotechnology B.V. De Brauwweg 13 3125 AE Schiedam The Netherlands Tel: +31 (0)10-2983555 Fax: +31 (0)10-4379648 E-mail: applikon@applikon.com ii

SAFETY SAFETY CLASS I EQUIPMENT This set of equipment has been designed in accordance with EN61010-1 "Safety Requirements for Electrical Equipment for Measurement, Control and Laboratory Use", and has been supplied in a safe condition. The Hardware manual contains information and warnings, which have to be followed by the user to ensure safe operation and to retain the equipment in safe condition. Before switching the equipment on, make sure that it is set to the line voltage. This equipment is designed for bioprocess control; it must not be used for other purposes! Caution, electrical shock hazard: Any interruption of the protective conductor inside or outside the apparatus or disconnection of the protective conductor terminal will make the apparatus dangerous. Intentional interruption is prohibited. Capacitors inside the apparatus may still be charged, even if the apparatus has been disconnected from all voltage sources. Any adjustment, maintenance and repair of the opened apparatus under voltage must be avoided as far as possible and, if inevitable, must only be carried out by qualified personnel. Make sure that only fuses with the required rated current and of the specified type (IEC 127) are used for replacement. The use of makeshift fuses and the short-circuiting of fuse holders are prohibited. Caution, electrical shock hazard: It is not permitted to connect equipment to the ez-control or the bioreactor without the qualification of double isolation or without the SELV (Separated Extra Low Voltage) qualification. iii

SAFETY Caution: In most cases, one or more pumps are installed in the ez-control. According to the European Community legislation on machinery, a pump drive is a machine with potential hazard. Only apply these drives with the pump heads and tubing that are described in this manual (chapter: pumps)! Do not use the tubing pump drives for other purposes than displacement of fluids (or gas). Switch off the power of the pump drive before replacing any tubes or pump heads. Application of damaged tubes may result in fluid leaking into the pump drive. Verify the tube quality inside the pump head before every fermentation run. Do not apply an Earth Leakage Circuit Breaker in the power supply of the Applikon control equipment (in an industrial environment or laboratory, an earth leakage circuit breaker is not mandatory). Because of the built-in main supply filters, an Earth Leakage Circuit Breaker may be activated. As a result, the power will be switched off! Additional electrical protection can be realized by installing a separation transformer between the mains and the control equipment. The ez-control weighs more than 18 kilos. Refer to chapter 1.6 of this manual. iv

CONFORMITY EU DECLARATION OF CONFORMITY The company Applikon Biotechnology B.V., residing in Schiedam, The Netherlands, hereby certifies that the instrument: ez-control for Autoclavable Bio Reactor Systems 1-20 liter meets the requirements of the following Directives of the European Union: - Directive 73/23/EEC on Low Voltage - Directive 89/336/EEC on Electromagnetic Compatibility; amendment 93/68/EC - Directive 98/37/EEC on Machinery ir A. Oudshoorn MBA January 5 th 2007 Applikon Biotechnology B.V. De Brauwweg 13 3125 AE Schiedam The Netherlands Tel.: +31 (0)10-2983555 Fax.: +31 (0)10-4379648 E-mail: applikon@applikon.com v

PUMP PROTECTION PROTECTION OF THE CIRCULATION PUMP If a Thermo Circulator is installed, it must be filled with tap water before the circulation pump can be started. Log in as Operator (refer to chapter Authorization of the Firmware Reference Manual). Make sure that the Thermo Circulator, the temperature control loop, and the circulation pump are switched off. Press the Temperature parameter button and the blue Thermo circulator button at the bottom of the option list. Connect the tap water supply to the Thermo Circulator, at the rear side of the ez-control. Press the Fill button (the button toggles between Fill and Stop filling) and watch the end of the drain tubing. As soon as water flows out of the drain tubing, press the Stop filling button. Now press the Activate button to start the Thermo Circulator. When the ez-control is loaded with firmware version 1.1x or higher, the heater-tank is automatically refilled on a regular basis. If the Thermo Circulator tank is filled with non-conductive water, an error message will be displayed. So make sure to use tap water to fill the tank. vi

CONTENTS TABLE OF CONTENTS SYMBOLS... ii SAFETY CLASS I EQUIPMENT... iii EU DECLARATION OF CONFORMITY... v PROTECTION OF THE CIRCULATION PUMP... vi 1 GENERAL... 1 1.1 Introduction... 1 1.2 Front View... 1 1.3 Emergency Stop Button... 2 1.4 Right-Side View... 2 1.5 Rear View... 4 1.6 Lifting the ez-control... 5 1.7 Sensor Input Specifications... 5 1.8 On/Off Actuators versus Continuous Actuators... 6 1.9 Actuator for Level / Foam Control... 6 1.10 Environmental Conditions... 7 1.11 Cleaning Instructions... 7 2 ACTUATORS... 8 2.1 Gas Supply Section... 8 2.1.1 Gas Selection Block... 8 2.1.2 Rotameters... 9 2.1.3 Mass Flow Controllers... 9 2.1.4 Valves in Gas Supply Lines... 9 2.2 Actuators for Temperature Control... 10 2.2.1 Heating Blanket... 10 2.2.2 Cold Water Valve and Thermo Circulator... 11 2.3 Pumps and Tubing... 12 2.3.1 How to Load the Tubing... 13 3 AGITATION... 14 3.1 Stirrer Speed and Torque Overview... 14 3.2 Stirrer Motors... 15 3.2.1 Motor P100... 15 3.2.2 Motor P140... 15 3.2.3 Motor P100, i=6... 15 3.2.4 Motor P310... 16 3.2.5 Motors P1000 and P1000, i=5... 16 3.2.6 Extended Power Supply Module for P1000 Motors... 16 3.3 Motor Cables... 16 3.4 Maintenance... 16 4 REACTORS... 17 4.1 Reactor Types... 17 4.1.1 1-Liter Reactor... 17 4.1.2 2-Liter Reactors... 18 4.1.3 3-Liter Reactors... 19 4.1.4 5-Liter Reactors... 20 4.1.5 7-Liter Reactors... 21 4.1.6 15-Liter Reactors... 22 4.1.7 20-Liter Reactor... 23 4.2 Cart for Large Reactors... 23 5 AUXILIARIES... 24 vii

CONTENTS 5.1 Stirrer Assemblies... 24 5.1.1 Top Stirrer Assemblies... 24 5.1.2 Impellers... 25 5.1.3 Baffles... 25 5.1.4 Calculations of Requirements... 26 5.2 Aeration... 28 5.2.1 Gas In-/Outlet Filter... 28 5.2.2 Spargers... 28 5.2.3 Air Overlay Assembly... 29 5.2.4 Pressure Relief Valve... 29 5.2.5 Air Outlet Condensor... 30 5.2.6 Tuning Valve... 30 5.3 Addition... 31 5.3.1 Septum Holder... 31 5.3.2 Addition Pipe... 31 5.3.3 Medium Inlet Triple... 31 5.3.4 Liquid Entry System... 32 5.3.5 Rapi-Lok Sterile Connector... 32 5.3.6 Liquid Addition Bottle... 32 5.4 Sampling and Drain... 33 5.4.1 Assembly Holder... 33 5.4.2 Sample Pipe... 33 5.4.3 Sample System... 35 5.5 Heat Exchangers... 36 5.6 Miscellaneous... 37 5.6.1 Sensor Holders... 37 5.6.2 Thermometer Pocket... 38 5.6.3 Blind Stoppers... 38 6 SENSORS... 39 viii

GENERAL 1.1 INTRODUCTION 1 GENERAL The Applikon autoclavable Bioreactor Systems basically consist of the following parts: an autoclavable Bioreactor with the appropriate auxiliaries like a stirrer assembly, sensors, baffles, an aeration assembly, etc. an ez-control Bio Controller for measurement and control of process variables (ph, temperature, DO, level and stirrer speed) with corresponding controller outputs in order to keep process conditions on set-point. The ez-control combines and supports actuators like pumps and valves in order to optimize the use of limited bench space. 1.2 FRONT VIEW Below, the ez-control Bio Controller is shown in combination with an autoclavable 3 liter jacketed Stirred Tank Reactor. Touch Screen Display Emergency Stop Button Stirrer Motor Pump Section Stirrer Assembly Gas Supply Section Rack for Reagent Bottles Bioreactor (Jacketed) 1

GENERAL 1.3 EMERGENCY STOP BUTTON If the Emergency Stop button is pressed, all actuators are switched off. The power supply to the sensor hardware and control device is maintained, so measuring the process parameters is continued. The calculated controller output however is not realized until the Emergency Stop situation is reset to normal. Reset an Emergency Stop situation by turning this key clockwise until it returns to the normal position. 1.4 RIGHT-SIDE VIEW The following four sensor connections can be found at the right-side panel: BNC connector for the ph sensor BNC connector for the DO sensor 3-pin audio connector for the Temperature sensor 2-pin audio connector for the Level sensor Below the sensor connections, two I/O connections are located: 5-pin audio connector for the Stirrer motor 8-pin audio connector for the Stirrer encoder 2

GENERAL If a Thermo Circulator is installed, three hydraulic connections and a tuning valve are located below the Rack for Reagent Bottles: The Quick Connector (with valve) Water to Reactor leads water from the Thermo Circulator to the jacket of the reactor or to the heat exchanger inside the reactor. The Quick Connector (without valve) Water from Reactor is the return for the Thermo Circulator. The Quick Connector (with valve) Water to Condenser leads cooling water to the condenser. The Quick Connector (without valve) Water from Condenser is the return for the condenser. The condensed water is transferred to the drain via the Quick Connector at the rear side of the cabinet. The Regulator Valve tunes the cooling water flow to the Condenser. All hose connectors are sized for 6 mm tubing. Temperature Control can also be achieved with a Heating Blanket and Cold Water Valve. In this case an extra electric connection for the Heating Blanket is present below the rack for reagent bottles. Heating Blankets are applied on single wall reactors. Cooling water is connected to the heat exchanger inside the reactor. 3

GENERAL 1.5 REAR VIEW The Power Section contains: A Euro socket for the mains power cable. A power switch. Four fuse holders; fuse rating: o 230 VAC: 4 x T 5A (5 x 20 mm). o 115 VAC: 2 x T 8A (electronics), 2 x T 6.3A (heater) (5 x 20 mm). Fuses must comply with IEC 127. The Communication Section contains the following connections: An RJ-45 Ethernet connection. A mini USB port. Two USB ports. A 9-pin sub-d connector. The Ethernet connection is used to communicate with a Supervisory Control And Data Acquisition (SCADA) program on a Host. Pin layout of the 9-pin sub-d connector: Pin Function Pin Function 1 Process Alarm (NC) 6 Deviation Alarm (NC) 2 Process Alarm (NO) 7 Deviation Alarm (NO) 3 Process Alarm (Common) 8 Deviation Alarm (Common) 4, 5, 9 Reserved for future use These alarm pins are potential-free. Connect a 24 VDC power supply with maximum current of 2 A. The gas inlets (carbon dioxide, oxygen, air and nitrogen) are Quick Connectors for tubing OD = 6 mm. Required inlet pressure for the gasses: 2 barg. In the lower part of the rear panel, two hydraulic connections (Quick Connectors) are located: Water inlet for Thermo Circulator or Cold Water Valve (with valve, hose barb 6 mm). Drain connection (without valve, hose barb 6 mm). The ez-control Bottle Rack for Reagent Bottles is optional. It is available in two versions: Z310113010 Bottle Rack for 1 to 7 liter reactors 3 x 0.5 liter bottles Z310113020 Bottle Rack for 15 to 20 liter reactors 1 x 0.5 & 2 x 1 liter bottles 4

GENERAL 1.6 LIFTING THE ez-control The ez-control weighs more than 18 kg. According to European legislation, lifting support is required for these devices (lift the device with two persons) Aa set of Lifting Handles is available for the ez-control: Z310113030 Set of Lifting Brackets for the ez-control. After unpacking the equipment, first mount the two handles: one at each side of the equipment. Note that the bottle rack has to be removed. After placing the equipment at its final location, the handles must be removed and the bottle rack can be mounted again. 1.7 SENSOR INPUT SPECIFICATIONS The applied sensor input amplifiers have the following specifications: ph amplifier: Range: 0 ph - 14 ph Accuracy: ±0.01 ph R in : > 10 15 Ω Temperature amplifier: Type: Pt-100 (3-wire type) Range: 0 o C - 150 o C Accuracy: ±0.1 o C DO amplifier: Type: Polarographic Range: 0 % - 500 % (air) 0 % - 100 % (oxygen) Accuracy: ±0.1 % Level amplifier: Type: On/Off signal Sensitivity: Firmware-selectable: High: Conductivity 26 µs equals Contact Low: Conductivity 200 µs equals Contact refer to section Additional Foam / Level Controller Settings of the Firmware Reference Manual. 5

GENERAL 1.8 ON/OFF ACTUATORS VERSUS CONTINUOUS ACTUATORS On/Off actuators like valves and pumps are controlled in a Pulse-Width Modulated manner: within a predefined cycle time, the output (Actuator Output, AO) is switched on during the On-time and off during the rest of the cycle. For example, if Cycle time = 20 sec. Duty cycle = 30 % then the On/Off Output will be repeatedly switched: on during 6 sec. off during 14 sec. 100 AO (%) 0 0 on off 6 20 Cycle Time on 26 off on T (sec.) Continuous actuators like mass flow controllers and stirrers do not have a cycle time or a duty cycle. Deviation from set-point results in a controller output that relates to the actuator output in a linear way. For example, if Controller Output = 30 % then the continuous output has an Actuator Output = 30 %. 100 AO (%) 30 0 0 30 CO (%) 100 1.9 ACTUATOR FOR LEVEL / FOAM CONTROL The actuator for the level / anti-foam controller is either off (AO = 0 %) or on (AO = 100 %). In most cases the desired situation = No Contact. When the status No Contact changes into Contact, first a Dead Time elapses after what the actuator is switched on during the Pulse Time interval. This sequence is repeated until the status changes into No Contact again. 100 AO (%) 0 No contact Dead Time Contact Pulse Time Dead Time T (sec.) Values for Dead Time and Pulse Time can be edited by using the Menu option Level Control Timing. The function of the level sensor (discrimination between no contact and contact ) can be selected by calibrating the level sensor. 6

GENERAL 1.10 ENVIRONMENTAL CONDITIONS The ez-control for Autoclavable Bioreactor Systems may be used at locations with the following environmental conditions: Indoor. Altitude: up to 2000 m. Temperature: 4 C to 45 C. Maximum relative humidity 80 % for temperatures up to 31 C, decreasing linearly to 50 % relative humidity at 45 C. Mains supply voltage: 115/230 VAC (+15%/-20%), 50/60 Hz. Transient over voltages according to INSTALLATION CATEGORIES II. POLLUTION DEGREE 2 in accordance with IEC 664. P-max = 1200 VA. 1.11 CLEANING INSTRUCTIONS The ez-control for Autoclavable Bioreactor Systems may be cleaned with a moist tissue in combination with a non-abrasive cleaner. 7

ACTUATORS 2 ACTUATORS The Actuator control part of the ez-control supports the following actuators: ph control: acid / base addition or aeration with carbon dioxide DO control: aeration with air, nitrogen or oxygen, stirrer speed control Temperature control: Thermo Circulator, Cold Water valve or Heating Blanket Level control: Level / Foam pump This chapter describes the applicable hardware (inside the ez-control cabinet and outside the bioreactor). 2.1 GAS SUPPLY SECTION The gas supply section supports (a combination of) a Gas Selection Block and up to four rotameters and/or four mass flow controllers. Gas Selection Block with up to four Gas Selection Valves 2.1.1 GAS SELECTION BLOCK The Gas Selection Block module offers easy switching (per gas) between gas overlay and sparging: upward position = overlay; downward position = sparging. In case no Gas Selection Block is applied, for each individual gas outlet a bulkhead union is mounted in the front. Spare holes are blinded. Rotameters Z311302020 Z310112040 Gas Selection Block Gas Outlet Bulkhead Connector Set Below, an example is shown on how to connect the gasses output of the ez-control to an air inlet sparger. All required articles are supplied in the start-up kit. Tubing Size 25 V0M5217225 Tubing Clamp V0N0000091 To Gas Sparger Gas Filter Z811302030 Reducer V0W0530004 Tubing Size 16 V0M5217000 8

ACTUATORS 2.1.2 ROTAMETERS Each rotameter contains a non-return valve at the gas outlet that prevents pressure differences to cause backflow through the rotameter. Rotameters (60 mm, direct reading scales): Z3RM002006 Rotameter 50 ml/min O 2, N 2, air Z3RM002011 Rotameter 100 ml/min O 2, N 2, air Z3RM002016 Rotameter 500 ml/min O 2, N 2, air Z3RM002020 Rotameter 1 l/min O 2, N 2, air Z3RM002025 Rotameter 5 l/min O 2, N 2, air Z3RM002030 Rotameter 10 l/min O 2, N 2, air Z3RM002035 Rotameter 50 l/min O 2, N 2, air Z3RM002041 Rotameter 50 ml/min CO 2 Z3RM002046 Rotameter 100 ml/min CO 2 Z3RM002051 Rotameter 500 ml/min CO 2 Z3RM002055 Rotameter 1 l/min CO 2 Z3RM002060 Rotameter 5 l/min CO 2 Z3RM002065 Rotameter 10 l/min CO 2 2.1.3 MASS FLOW CONTROLLERS Behind the rotameter panel, inside the gas supply section, up to four mass flow controllers (MFC) can be mounted. The MFC comes with a mounting set (mounting material, connectors and tubing). It also contains a non-return valve at the gas outlet that prevents pressure differences to cause backflow through the MFC. The Controller Output to the MFC is communicated digitally (RS485 protocol). Z310112020 Mass Flow Controller 0-30 SL/min 2.1.4 VALVES IN GAS SUPPLY LINES At the inner rear side of the gas supply box, per gas a solenoid valve can be mounted before the rotameter or mass flow controller. Specifications for the gas inlet lines: Gas inlet connection: bulkhead union 6 mm Gas outlet connection: bulkhead union 6 mm Required inlet pressure: 2 barg Z310112010 Solenoid Valve Assembly 24 V 9

ACTUATORS 2.2 ACTUATORS FOR TEMPERATURE CONTROL For temperature control several actuator configurations can be used, as presented in the following table. Option Actuator for Heating Actuator for Cooling 1 Heating Blanket None (temperature loss to environment) 2 Heating Blanket Cold Water Valve 3 Thermo Circulator Thermo Circulator 2.2.1 HEATING BLANKET A heating blanket is wrapped around a glass bioreactor (no jacket). Power consumption: 1 liter: 73 W 2 liter: 100 W 3 liter: 110 W 5/7 liter: 178 W 15/20 liter: 383 W The maximum temperature of a heating blanket is 80 C. The heating blanket is connected to the 3-pins audio connector (right side panel of the ez-control cabinet). DANGER! ELECTRICAL SHOCK HAZARD: when the actuator is switched on, main voltage is directed to the connector of the heating blanket! DO NOT disconnect the heating blanket without switching OFF the Temperature Control Loop! Make sure that the heating blanket is NOT manually activated! Heating Blankets: Z311020010 Heating blanket 230 VAC for 1 liter dished bioreactor Z311020022 Heating blanket 230 VAC for 2 liter dished bioreactor Z311020030 Heating blanket 230 VAC for 3 liter dished bioreactor Z311020072 Heating blanket 230 VAC for 5 and 7 liter dished bioreactor Z311020150 Heating blanket 230 VAC for 15 and 20 liter dished bioreactor Z310111020 Heating blanket module 115 VAC, 200 W; 230 VAC, 400 W Z310111050 Heating blanket module 115 VAC, 400 W 10

ACTUATORS 2.2.2 COLD WATER VALVE AND THERMO CIRCULATOR If a Cold Water Valve assembly or a Thermo Circulator is used for temperature control, hydraulic connections must be made between bioreactor and ez-control cabinet: Connectors for Water Inlet and Atmospheric Drain are placed at the lower rear part of the ez-control cabinet. Hose barb connections: OD = 6 mm. The side panel of cabinet contains the Quick Connectors for direction of water to and from the reactor (jacket or heat exchanger) and the condenser. Quick Connector Hose barb: 6 mm. Schematic presentation of the Thermo Circulator: Tap water must be used as medium for heating and cooling (do not use d.i.-water or organic solvents)! If no condenser connection and/or cold water valve / thermo circulator are applied, the mounting positions for the Quick Connectors are covered with blind stoppers. The Regulator Valve is used to tune the water flow to the condenser. Z310111010 Thermo Circulator Module 230 VAC Z310111015 Thermo Circulator Module 115 VAC Z310111031 Cold Water Valve Assembly with Condenser Regulator Valve Z310111040 Condenser Regulation Valve (without Cold Water Valve Assembly) 11

ACTUATORS 2.3 PUMPS AND TUBING At the Pump Section up to three tubing pump drives with pump heads may be installed. The figure shows the default pump configuration. CAUTION! According to the European Community legislation on machinery, a pump drive is a machine with potential hazard. Only apply these drives with the pump heads and tubing that are described in this chapter. Do not use the tubing pump drives for other purposes than displacement of fluids (or gas). RISK OF PHYSICAL INJURY! Switch off the power of the pump drive before replacing any tubes or pump heads. Application of damaged tubes may result in fluid leaking into the pump drive. Verify the tube quality inside the pump head before every fermentation run. Fixed-speed pump drives for ez-control: Z310116060 Pump drive 20 rpm with Pump head package 102R (standard) Z310116050 Pump drive 20 rpm with Pump head package 313D (advanced) Stand-Alone Pumps (with 230 VAC power supply): Z375548500 Eco Drive variable speed: 7-200 rpm for short-term operation Z377521570 Uni Drive variable speed: 1-100 rpm for continuous operation Z375237000 Drive variable speed: 1-100 rpm with digital display For specific information concerning the stand-alone pumps and pump heads, refer to the Masterflex User Manuals that come with the pumps. Tubing: Z364111300 Silicone tubing type 13 7.5 m ID = 0.8 mm 0.06 ml/rev Z364111400 Silicone tubing type 14 7.5 m ID = 1.6 mm 0.2 ml/rev Z364111600 Silicone tubing type 16 7.5 m ID = 3.1 mm 0.8 ml/rev Z364112500 Silicone tubing type 25 7.5 m ID = 4.8 mm 1.7 ml/rev Z364111800 Silicone tubing *) type 18 7.5 m ID = 7.9 mm 3.8 ml/rev Z364021300 Norprene food tubing type 13 15 m ID = 0.8 mm 0.06 ml/rev Z364021400 Norprene food tubing type 14 15 m ID = 1.6 mm 0.2 ml/rev Z364021600 Norprene food tubing type 16 15 m ID = 3.1 mm 0.8 ml/rev Z364022500 Norprene food tubing type 25 15 m ID = 4.8 mm 1.7 ml/rev Z364021800 Norprene food tubing *) type 18 15 m ID = 7.9 mm 3.8 ml/rev *) Only applicable in pump type 313D. 12

ACTUATORS This example shows how to add liquids via a pump and the medium inlet triple. All required articles are supplied in the start-up kit. Tubing Size 14 Z362041400 Tubing Size 14 Z362041400 Tubing Clamp V0N0000091 Tubing Clamp V0N0000093 Tubing Size 25 V0M5217225 Reducer V0W0530005 Addition Bottle Z811302009 2.3.1 HOW TO LOAD THE TUBING Pump head type 102R: RISK OF PHYSICAL INJURY! Pump head 102R must be started manually. Carefully load the tubing in the pump head: watch your fingers! Pump head type 313D (advanced): 13

AGITATION 3 AGITATION This chapter describes several DC permanent magnet motors for stirrer assemblies for Applikon bioreactors. An encoder, present in the motor, is used for feedback to the motor controller. The stirrer motor comes with four motor studs to fit in the stirrer assembly and a covered (flexible) coupling fork to avoid noise and vibrations during operation. No tools are required to mount the stirrer motor on top of the stirrer assembly. Warning: The stirrer motor is a machine with potential hazard (moving parts). In order to avoid dangerous situations, make sure that the stirrer motor is only operated when it is properly seated in the stirrer assembly of the Applikon autoclavable bioreactor. The stirrer motor must only be used as part of a Bioprocess (bioreactor with control system). It therefore comes with supplier s declaration of conformity of type IIb regarding the European Community legislation on machinery. The stirrer motor must not be put into service until the machinery into which it is to be incorporated has been declared to conform to machinery directive 98/37/EC. 3.1 STIRRER SPEED AND TORQUE OVERVIEW The stirrer speed range and maximum torque for the different motor types is listed below. Reactor Volume (liter) Maximum torque (Nm) Default speed range (rpm) 2 to 7 0.20 0 to 1250 P100 2 to 7 0.30 0 to 800 P140 2 to 7 0.55 0 to 1250 P310 Motor type 15 to 20 1.0 0 to 200 P100, i=6 15 to 20 3.0 0 to 1000 P1000 15 to 20 12 0 to 200 P1000, i=5 The specifications of each motor type are listed in the following sections. 14

AGITATION 3.2 STIRRER MOTORS 3.2.1 MOTOR P100 Standard motor for the Applikon 2 and 3 liter bioreactors with low viscous media at stirrer speeds of 0 to 1250 rpm. The motor can also be used with the 1 to 7 liter cell cultures with lower stirrer speeds, between 100 and 200 rpm. Maximum torque: 0.20 Nm. Weight: 1.6 kg. Includes: motor studs and flexible coupling. The motor studs fit in the Top Stirrer Assemblies of the 2 to 7 liter bioreactors. Z510000010 Stirrer Motor Assembly P100 3.2.2 MOTOR P140 Heavy duty motor for the Applikon 5 and 7 liter bioreactors, used in tough applications with viscous media and stirrer speeds up to 800 rpm. Maximum torque: 0.30 Nm. Weight: 1.9 kg. Includes: motor studs and flexible coupling. The motor studs fit in the Top Stirrer Assemblies of the 2 to 7 liter bioreactors. Z510000020 Stirrer Motor Assembly P140 3.2.3 MOTOR P100, I=6 Standard motor for cell culture bioreactors and a standard stirrer speed range of 0 to 200 rpm. Maximum torque: 1.0 Nm. Weight: 2.4 kg. Includes: motor studs and coupling. Stirrer speed is reduced 6.25 times by a planetary gearbox. The motor studs fit in the Top Stirrer Assemblies of the 15 and 20 liter bioreactors. Z510000011 Stirrer Motor Assembly P100, i=6 15

AGITATION 3.2.4 MOTOR P310 Upgrade motor for the 5 and 7 liter Applikon autoclavable bioreactors, to be used in normal applications with stirrer speeds up to 1250 rpm. Maximum torque: 0.55 Nm. Weight: 3.9 kg. Includes: motor studs and flexible coupling. The motor studs fit in the Top Stirrer Assemblies of the 2 to 7 liter bioreactors. Z510000030 Stirrer Motor Assembly P310 3.2.5 MOTORS P1000 AND P1000, I=5 Standard motor for the Applikon autoclavable 15 and 20 liter bioreactors with a stirrer speed range of 0 to 1000 rpm. The P1000 can also be used in the in-situ sterilizable bioreactors as a top-drive stirrer. Maximum torque: 3.0 Nm. Weight: 8.1 kg. Includes: motor studs and coupling. The motor studs fit in the Top Stirrer Assemblies of the 15 and 20 liter bioreactors. The P1000, i=5 motor is based on the P1000 motor, but a planetary gearbox reduces the stirrer speed 5 times to a range of 0 to 200 rpm. Maximum torque: 12 Nm. Weight: 11.4 kg. Z510000040 Stirrer Motor Assembly P1000 Z510000041 Stirrer Motor Assembly P1000, i=5 3.2.6 EXTENDED POWER SUPPLY MODULE FOR P1000 MOTORS Z310110050 3.3 MOTOR CABLES Z510121011 Z510121012 Z510121020 3.4 MAINTENANCE Power Supply Upgrade P1000 Motor (required to enable the maximum motor torque) Motor Cable for Stirrer P100 & P140 Motor Cable for Stirrer P310 & P1000 Encoder Cable The stirrer motor is virtually maintenance-free. The only parts that require regular maintenance are the carbon brushes and the ball bearings of the motor. The life span of the carbon brushes depends on the operating conditions (normally approximately 10,000 operating hours). Minimum length of the carbon brush: 9 mm. When the carbon brushes are replaced, it is advised also to replace the ball bearings of the motor. Operating the stirrer motor with worn-out carbon brushes will cause irreversible damage! 16

REACTORS 4 REACTORS This chapter describes the autoclavable range of reactors and their head plate specifications. 4.1 REACTOR TYPES Materials in contact with the medium (all reactors): borosilicate glass stainless steel silicone rubber optional: viton or EPDM 4.1.1 1-LITER REACTOR Reactor type 1 liter, dished bottom Inner diameter 95 mm Inner height (maximum) 200 mm Liquid height (working volume) 150 mm Required autoclave space (H x D) 350 x 175 mm Overall height reactor 275 mm Total volume 1.25 liter Working volume 0.9 liter Minimum working volume 0.3 liter H/D total 2.0 H/D working volume 1.5 Ports in head plate: 1 * M30 x 1 1 * G3/4" 4 * M18 x 1.5 The 1-liter reactor has the following connections welded in the head plate: Water inlet and outlet (heat exchanger) Sample pipe Air inlet pipe Inoculation pipe Air outlet pipe Thermometer pocket Z611000110 1-liter Dished-bottom Reactor 17

REACTORS 4.1.2 2-LITER REACTORS Reactor type 2 liter, dished bottom Inner diameter 105 mm Inner height (maximum) 240 mm Liquid height (working volume) 156 mm Required autoclave space (H x D) 400 x 200 mm Overall height reactor 290 mm Total volume 2.2 liter Working volume 1.7 liter Minimum working volume 0.3 liter H/D total 2.3 H/D working volume 1.9 Ports in head plate: 1 * M30 x 1 1 * G3/4" 5 * M18 x 1.5 3 * 6 mm 6 * 10 mm Z611000210 2-liter Dished-bottom Reactor Reactor type 2 liter, jacketed Inner diameter 105 mm Inner height (maximum) 240 mm Liquid height (working volume) 156 mm Required autoclave space (H x D) 400 x 200 mm Overall height reactor 290 mm Total volume 2.2 liter Working volume 1.7 liter Minimum working volume 0.3 liter H/D total 2.3 H/D working volume 1.9 Jacket volume 1.3 liter Ports in head plate: 1 * M30 x 1 1 * G3/4" 5 * M18 x 1.5 3 * 6 mm 6 * 10 mm Z611000220 2-liter Jacketed Reactor 18

REACTORS 4.1.3 3-LITER REACTORS Reactor type 3 liter, dished bottom Inner diameter 130 mm Inner height (maximum) 250 mm Liquid height (working volume) 200 mm Required autoclave space (H x D) 400 x 200 mm Overall height reactor 290 mm Total volume 3.1 liter Working volume 2.7 liter Minimum working volume 0.6 liter H/D total 1.9 H/D working volume 1.5 Ports in head plate: 1 * M30 x 1 1 * G3/4" 5 * M18 x 1.5 3 * 6 mm 6 * 10 mm 2 * 12 mm Z611000310 3-liter Dished-bottom Reactor Reactor type 3 liter, jacketed Inner diameter 130 mm Inner height (maximum) 250 mm Liquid height (working volume) 200 mm Required autoclave space (H x D) 400 x 230 mm Overall height reactor 290 mm Total volume 3.1 liter Working volume 2.7 liter Minimum working volume 0.6 liter H/D total 1.9 H/D working volume 1.5 Jacket volume 1.2 liter Ports in head plate: 1 * M30 x 1 1 * G3/4" 5 * M18 x 1.5 3 * 6 mm 6 * 10 mm 2 * 12 mm Z611000320 3-liter Jacketed Reactor 19

REACTORS 4.1.4 5-LITER REACTORS Reactor type 5 liter, dished bottom Inner diameter 160 mm Inner height (maximum) 250 mm Liquid height (working volume) 180 mm Required autoclave space (H x D) 400 x 200 mm Overall height reactor 290 mm Total volume 4.8 liter Working volume 3.4 liter Minimum working volume 0.9 liter H/D total 1.6 H/D working volume 1.1 Ports in head plate: 1 * M30 x 1 1 * G3/4" 5 * M18 x 1.5 10 * 10 mm 2 * 12 mm Z611000510 5-liter Dished-bottom Reactor Reactor type 5 liter, jacketed Inner diameter 160 mm Inner height (maximum) 250 mm Liquid height (working volume) 180 mm Required autoclave space (H x D) 450 x 260 mm Overall height reactor 330 mm Total volume 4.8 liter Working volume 3.4 liter Minimum working volume 0.9 liter H/D total 1.6 H/D working volume 1.1 Jacket volume 1.8 liter Ports in head plate: 1 * M30 x 1 1 * G3/4" 5 * M18 x 1.5 10 * 10 mm 2 * 12 mm Z611000520 5-liter Jacketed Reactor 20

REACTORS 4.1.5 7-LITER REACTORS Reactor type 7 liter, dished bottom Inner diameter 160 mm Inner height (maximum) 350 mm Liquid height (working volume) 270 mm Required autoclave space (H x D) 510 x 260 mm Overall height reactor 390 mm Total volume 6.8 liter Working volume 5.4 liter Minimum working volume 1.5 liter H/D total 2.2 H/D working volume 1.8 Ports in head plate: 1 * M30 x 1 1 * G3/4" 5 * M18 x 1.5 10 * 10 mm 2 * 12 mm Z611000710 7-liter Dished-bottom Reactor Reactor type 7 liter, jacketed Inner diameter 160 mm Inner height (maximum) 350 mm Liquid height (working volume) 270 mm Required autoclave space (H x D) 540 x 260 mm Overall height reactor 425 mm Total volume 6.8 liter Working volume 5.4 liter Minimum working volume 1.5 liter H/D total 2.2 H/D working volume 1.8 Jacket volume 2.4 liter Ports in head plate: 1 * M30 x 1 1 * G3/4" 5 * M18 x 1.5 10 * 10 mm 2 * 12 mm Z611000720 7-liter Jacketed Reactor 21

REACTORS 4.1.6 15-LITER REACTORS Reactor type 15 liter, dished bottom Inner diameter 220 mm Inner height (maximum) 440 mm Liquid height (working volume) 320 mm Required autoclave space (H x D) 800 x 400 mm Overall height reactor 520 mm Total volume 15.5 liter Working volume 12 liter Minimum working volume 3.0 liter H/D total 2.0 H/D working volume 1.45 Ports in head plate: 1 * 69 mm 6 * 27 mm 2 * 12 mm 10 * 10 mm Z611001510 15-liter Dished-bottom Reactor Reactor type 15 liter, jacketed Inner diameter 240 mm Inner height (maximum) 438 mm Liquid height (working volume) 274 mm Required autoclave space (H x D) 800 x 500 mm Overall height reactor 520 mm Total volume 18.2 liter Working volume 12 liter Minimum working volume 3.0 liter H/D total 1.8 H/D working volume 1.15 Jacket volume 7.4 liter Ports in head plate: 1 * 69 mm 6 * 27 mm 2 * 12 mm 10 * 10 mm Z611001520 15-liter Jacketed Reactor 22

REACTORS 4.1.7 20-LITER REACTOR Reactor type 20 liter, dished bottom Inner diameter 220 mm Inner height (maximum) 620 mm Liquid height (working volume) 425 mm Required autoclave space (H x D) 1000 x 400 mm Overall height reactor 715 mm Total volume 22 liter Working volume 16 liter Minimum working volume 3.0 liter H/D total 2.8 H/D working volume 1.9 Ports in head plate: 1 * 69 mm 6 * 27 mm 2 * 12 mm 10 * 10 mm Z611002010 20-liter Dished-bottom Reactor 4.2 CART FOR LARGE REACTORS Since moving a filled 15- or 20-liter reactor is not an easy job, a special cart has been developed to carry the three largest reactor types. The inner holes are designed to accept the tripod of the dished-bottom reactors; the outer holes fit the 15-liter jacketed reactor assembly. Dimensions: h = 960 mm w = 350 mm d = 460 mm Material: Frame: stainless steel 316 Wheels: duro-plastic (autoclavable material) Z811110001 Cart for the 15- and 20-liter reactors 23

AUXILIARIES 5 AUXILIARIES This chapter describes the head plate auxiliaries, i.e. devices that can be mounted in a head plate and protrude into the bioreactor. 5.1 STIRRER ASSEMBLIES For agitation, stirrer assemblies, impellers and baffles can be applied. Use the formulas in section 5.1.4 to determine which devices are optimal for your application. 5.1.1 TOP STIRRER ASSEMBLIES All stirrer assemblies are mounted at the central stirrer port in the bioreactor head plate. Lipseal Stirrer Assemblies for 1 to 7 liter reactors: Z81315R002 Lipseal Stirrer Assembly 1 liter Z81315R003 Lipseal Stirrer Assembly 2, 3, 5 liter Z81315R007 Lipseal Stirrer Assembly 7 liter Lipseal Stirrer Assemblies for 15 and 20 liter reactors: Z81315R010 Lipseal Stirrer Assembly 15 liter Z81315R020 Lipseal Stirrer Assembly 20 liter Magnetically Coupled Stirrer Assembly for 1 to 7 liter reactors (shown without stirrer shaft): Z81315MG02 MC Stirrer Assembly 1 liter Z81315MG03 MC Stirrer Assembly 2, 3, 5 liter Z81315MG07 MC Stirrer Assembly 7 liter Magnetically Coupled Stirrer Assembly for 15 and 20 liter reactors: Z81315MG10 MC Stirrer Assembly 15 liter Z81315MG20 MC Stirrer Assembly 20 liter 24

AUXILIARIES 5.1.2 IMPELLERS All impellers fit to the 8 mm and 15 mm diameter shafts of the stirrer assemblies. Turbine impellers: Z81312RS02 Rushton impeller 4 blades OD = 46 mm 1, 2, 3 liter Z81313R602 Rushton impeller 6 blades OD = 46 mm 1, 2, 3 liter Z81313R645 Rushton impeller 6 blades (vortex) OD = 45 mm 1, 2, 3 liter Z81313R607 Rushton impeller 6 blades OD = 60 mm 5, 7 liter Z81313R610 Rushton impeller 6 blades OD = 75 mm 15, 20 liter Z81313R611 Rushton impeller 6 blades (vortex) OD = 75 mm 15, 20 liter Marine impellers: Z81314RC02 Marine impeller vortex OD = 45 mm 1, 2, 3 liter Z81314RC03 Marine impeller scoping OD = 45 mm 1, 2, 3 liter Z82314RC07 Marine impeller vortex OD = 60 mm 5, 7 liter Z82314RC08 Marine impeller scoping OD = 60 mm 5, 7 liter Z82314RC10 Marine impeller vortex OD = 74 mm 15, 20 liter Z82314RC11 Marine impeller scoping OD = 74 mm 15, 20 liter 5.1.3 BAFFLES Baffles are used to increase the mixing efficiency. Without baffles, the medium flow can become laminar, causing poor mixing efficiency and mass transfer. For optimal mixing performance, the baffles are mounted in the head plate, near the reactor wall. Applicable number of baffles: 1 to 3. The 1-liter reactor comes with two baffles that are fixed to the heat exchanger. The baffle assembly consists of one baffle and mounting material. Z81326KS03 Baffle Assembly 2 and 3 liter 6 mm port Z81326KS05 Baffle Assembly 5 liter 10 mm port Z81326KS07 Baffle Assembly 7 liter 10 mm port Z81326KS10 Baffle Assembly 15 liter 10 mm port Z81326KS20 Baffle Assembly 20 liter 10 mm port 25

AUXILIARIES 5.1.4 CALCULATIONS OF REQUIREMENTS 5.1.4.1 POWER REQUIRED FOR STIRRING The required power of the stirrer motor depends on the number, type and diameter of the impellers, on the density of the medium, and on the stirrer speed. The required power (in Watt) per impeller of a stirrer motor in non-aerated media is given by the equation: 3 N 5 P = ρ D Np 60 where: P = the required power of the stirrer motor (W) ρ = the density of the medium (kg/m 3 ) N = the stirrer speed (rpm) D Np = the impeller diameter (m) = the power number of the impeller type: Rushton turbine impellers: Np = 6 ADI marine impellers: Np = 1.5 When mounted according to the given configuration, a second or third impeller on a shaft requires only 90 % of the power of the first impeller. This means that the equation above is multiplied with the factor 1.9 for two impellers and with a factor 2.8 for three impellers. 5.1.4.2 REQUIRED TORQUE The required torque (in Nm) of the stirrer motor is related to its power according to the following equation: 60 P M = 2 π N where: M = the required torque of the stirrer motor (Nm) P = the power of the stirrer motor (W) N = the stirrer speed (rpm) 5.1.4.3 IMPELLER TIP SPEED The Tip Speed (in m/s) of an impeller at a certain stirrer speed is given by the equation: N T = π D 60 where: T = the impeller tip speed (m/s) N = the stirrer speed (rpm) D = the impeller diameter (m) Section 5.1.4.4 shows examples of calculations of required power, torque, and impeller tip speed. Section 3.1 specifies the maximum torque that can be supplied by each of the available stirrer motors. 26

AUXILIARIES 5.1.4.4 EXAMPLES Required stirrer power, torque and corresponding tip speed for non-aerated media with a density of 1100 kg/m 3 are calculated as follows: 1, 2 or 3 liter reactor with two 6-bladed turbine impellers of 45 mm, at 1250 rpm: required power: P = 1100 * (1250 / 60) 3 * (0.045) 5 * 6 * 1.9 21 W required torque: M = 21 * 60 / (2 * π * 1250) 0.16 Nm tip speed: T = (1250 / 60) * π * 0.045 2.9 m/s 5 or 7 liter reactor with two turbine impellers of 60 mm, at 800 rpm: required power: P = 1100 * (800 / 60) 3 * (0.060) 5 * 6 * 1.9 23 W required torque: M = 23 * 60 / (2 * π * 800) 0.28 Nm tip speed: T = (800 / 60) * π * 0.060 2.5 m/s 15 liter reactor with two turbine impellers of 75 mm, at 800 rpm: required power: P = 1100 * (800 / 60) 3 * (0.075) 5 * 6 * 1.9 70 W required torque: M = 70 * 60 / (2 * π * 800) 0.85 Nm tip speed: T = (800 / 60) * π * 0.075 3.1 m/s 20 liter reactor with three turbine impellers of 75 mm, at 800 rpm: required power: P = 1100 * (800 / 60) 3 * (0.075) 5 * 6 * 2.8 105 W required torque: M = 105 * 60 / (2 * π * 800) 1.25 Nm tip speed: T = (800 / 60) * π * 0.075 3.1 m/s 15 liter reactor with one marine impeller of 74 mm, at 200 rpm: required power: P = 1100 * (200 / 60) 3 * (0.15) 5 * 1.5 0.14 W required torque: M = 0.14 * 60 / ( 2 * π * 200) 0.065 Nm tip speed: T = (200 / 60) * π * 0.074 0.8 m/s In bacterial cultures with aeration of approximately 2 VVM (2 gas volumes per reactor (working) volume per minute), the required motor power and torque will show a substantial decrease in relation to the calculated value. Friction in the (ball) bearings may cause power losses of 10 % to 20 %. 27

AUXILIARIES 5.2 AERATION 5.2.1 GAS IN-/OUTLET FILTER The bacterial air filter is an economical filter for sterile gas delivery and venting applications. The hydrophobic PTFE filter membrane excludes the risk of contamination. The filter is autoclavable. For venting applications, a condenser must be applied in order to prevent the filter from becoming clogged with water. Housing material: polypropylene Effective filtration area: 25 cm 2 Pore size: 0.2 μm Typical flow rate: 40 liter/minute at 0.4 barg (6 psig) Hose barb connection: Stepped hose barbs: 7 / 9.5 mm Maximum temperature: 132 C Z811302030 Disposable air filter 5.2.2 SPARGERS Spargers or gas inlet pipes are tubes that are immersed in the medium. Consequently gas that is added is sparged in the medium. 5.2.2.1 L-TYPE SPARGER To meet the oxygen demand of a culture, a sterile gas stream can be sparged through the culture, using an air-inlet pipe. This pipe can be applied when high gas flow rates are required, since this pipe causes hardly any pressure drop. The holes in this pipe are located at the bottom to make sure that medium will be driven out by the gas stream. The 1 liter reactor comes with an L-type sparger that is fixed in the head plate. Z81318L002 Sparger L-type 2 liter 10 mm port Z81318L003 Sparger L-type 3 liter 10 mm port Z81318L005 Sparger L-type 5 liter 10 mm port Z81318L007 Sparger L-type 7 liter 10 mm port Z81318L010 Sparger L-type 15 liter 10 mm port Z81318L020 Sparger L-type 20 liter 10 mm port 28

AUXILIARIES 5.2.2.2 POROUS SPARGER In cell culture cultivations high gas flow will damage the cells due to shear forces. To meet the oxygen demand of the cells at lower gas flows the exchange surface must be high. This is achieved by using a porous sparger with a pore size of 15 µm. This sparger produces tiny gas bubbles for optimum gas distribution. Use the Ceramic Sparger to increase the Oxygen Transfer Rate (OTR): oxygen transfer (k L a-value) of the ceramic sparger is about 3 times higher than the transfer with the porous sparger. The ceramic sparger must not be used with serum-containing media (foam formation!). Porous Sparger with sintered Metal Tip: Z81318L004 Sparger Porous 2 liter 10 mm port Z811303005 Sparger Porous 3 liter 10 mm port Z81318L006 Sparger Porous 5 liter 10 mm port Z81318L008 Sparger Porous 7 liter 10 mm port Z81318L011 Sparger Porous 15 liter 10 mm port Z811303008 Sparger Porous for air-inlet pipe Porous Sparger with Ceramic Tip: Z81318CS03 Sparger Ceramic 3 liter incl. Gas Inlet pipe Z81318CS07 Sparger Ceramic 7 liter incl. Gas Inlet pipe Z81318CS15 Sparger Ceramic 15 liter incl. Gas Inlet pipe Z81318CS00 Ceramic tip for sparger 3, 7, and 15 liter 5.2.3 AIR OVERLAY ASSEMBLY Air overlay means head space aeration (separate from, or in combination with, sparging gas through the culture). This assembly can be used for either gas overlay or gas outlet. Z81308LU02 Air overlay assembly 2 to 20 liter 10 mm port 5.2.4 PRESSURE RELIEF VALVE When over-pressure is applied in the (glass) Applikon reactors, it is advised to install a relief valve. The relief valve opens at a pressure of 0.5 barg. Z811302050 Pressure relief valve 1 to 7 liter M18 x 1.5 port Z811302060 Pressure relief valve 15 and 20 liter 27 mm port 29

AUXILIARIES 5.2.5 AIR OUTLET CONDENSOR Working at elevated temperatures and using aeration of the culture might cause too much evaporation during fermentation, causing an increase of nutrient concentration and a decrease in volume. An air-outlet condenser can prevent this. Air-outlet condensers (SS = Stainless Steel): Z81308L003 Glass 1 to 5 liter 12 mm ph nipple Z81308L002 SS 1 to 5 liter M18 x 1.5 port Z81308L007 SS 7 liter M18 x 1.5 port Air-outlet condensers (SS = Stainless Steel): Z81308L010 SS 15, 20 liter 27 mm port Z81308L011 SS, baffled 15, 20 liter 27 mm port 5.2.6 TUNING VALVE The tuning valve can be installed on top of the stainless steel air outlet condenser in order to create a small over-pressure in the reactor. This has the following advantages: Risk of contamination is reduced. Oxygen transfer to the medium is increased. Sampling the culture is eased. For safety reasons it is strongly advised to use this tuning valve in combination with the pressure relief valve (see section 5.2.4). Z811302020 Tuning valve for 1 to 20 liter reactor 30

AUXILIARIES 5.3 ADDITION During preparation and while running a process, fluids will be added to the reactor for medium addition, inoculation, ph and level control, perfusion, etc. 5.3.1 SEPTUM HOLDER The septum holder is equipped with a silicone rubber septum and can be used as a universal addition port by piercing it with one or more needles. The needle can be used to add a fluid or gas to the culture. Z81302PD02 Septum Holder 1 to 7 liter M18 x 1.5 port Z81302PD10 Septum Holder 15 and 20 liter 27 mm port Z81309IN02 Needle for septum 5.3.2 ADDITION PIPE The addition pipe can be used to add fluids (or gasses) to the reactor. Z81324MT02 Addition pipe ID = 4 mm 10 mm port Z81324MT05 Addition pipe ID = 4 mm 12 mm port 5.3.3 MEDIUM INLET TRIPLE The medium inlet triple allows you to equip one head plate port with three additions (e.g. for acid, alkali and anti-foam addition). This device can be used to expand the number of entries beyond the number of ports in the head plate. Z81324MT03 Inlet triple 1 to 7 liter M18 x 1.5 port Z81324MT10 Inlet triple 15 and 20 liter 27 mm port 31

AUXILIARIES 5.3.4 LIQUID ENTRY SYSTEM When running a continuous culture, back growth of organisms into the medium container must be prevented. The liquid entry system uses a sterile gas flow to transfer the fresh medium to the reactor. In this way, direct contact between the culture and the medium storage container does not exist. The liquid entry system fits into the ph sensor nipple. Z81309IN03 Liquid entry system 12 mm ph sensor nipple 5.3.5 RAPI-LOK STERILE CONNECTOR The autoclavable Rapi-Loks are a fast and reliable way to make or break tubing connections during a fermentation or cell culture process. Materials: Body and blind caps: SS 316L O-rings: silicone Z81324MT50 Rapi-Lok male coupling bore size (ID) 2 mm Z81324MT51 Rapi-Lok female coupling bore size (ID) 2 mm Z81324MT52 Rapi-Lok male coupling bore size (ID) 4 mm Z81324MT53 Rapi-Lok female coupling bore size (ID) 4 mm Z81324MT54 Rapi-Lok male coupling bore size (ID) 6 mm Z81324MT55 Rapi-Lok female coupling bore size (ID) 6 mm Z81324MT56 Rapi-Lok blind cap for male coupling Z81324MT57 Rapi-Lok blind cap for female coupling 5.3.6 LIQUID ADDITION BOTTLE The liquid addition bottle comes with an air-inlet filter. Z811302009 Liquid Addition Bottle 0.5 liter Z811302010 Liquid Addition Bottle 1.0 liter Z811302011 Liquid Addition Bottle 2.0 liter Z811302012 Liquid Addition Bottle 5.0 liter Z811302013 Liquid Addition Bottle 10 liter Z811302014 Liquid Addition Bottle 20 liter 32

AUXILIARIES 5.4 SAMPLING AND DRAIN The 1 liter reactor comes with a sample pipe that is fixed in the head plate. 5.4.1 ASSEMBLY HOLDER The Assembly Holder accepts any 6 mm (OD) tube. The insertion length of the tube can be varied, so that additions to or sampling from the culture fluid can take place at any level inside the reactor. Z811302015 Assembly Holder 2 to 7 liter M18 x 1.5 port Z81320AH00 Assembly Holder 2 to 20 liter 10 mm port 5.4.2 SAMPLE PIPE 5.4.2.1 FIXED-LENGTH SAMPLE PIPE ASSEMBLY This assembly is used to sample the culture fluid. Tube diameter (OD): 6 mm fits in a 10 mm port 9.5 mm fits in a 12 mm port Z81319MB03 Sample Pipe fixed-length 2 to 5 liter 10 mm port Z81319MB05 Sample Pipe fixed-length 2 to 5 liter 12 mm port Z81319MB07 Sample Pipe fixed-length 7 liter 10 mm port Z81319MB08 Sample Pipe fixed-length 7 liter 12 mm port Z81319MB10 Sample Pipe fixed-length 15 liter 10 mm port Z81319MB12 Sample Pipe fixed-length 15 liter 12 mm port Z81319MB20 Sample Pipe fixed-length 20 liter 10 mm port 5.4.2.2 HEIGHT-ADJUSTABLE SAMPLE PIPE This pipe is used to sample the culture fluid at any desired level. Tube diameter (ID): 4 mm. The assembly consists of: An assembly holder for 6 mm tubes; An OD 6 mm sample tube. Z81319MB04 Sample Pipe height-adjustable 2 to 5 liter 10 mm port Z81319MB06 Sample Pipe height-adjustable 7 liter 10 mm port Z81319MB21 Sample Pipe height-adjustable 15 liter 10 mm port Z81319MB25 Sample Pipe height-adjustable 20 liter 10 mm port 33

AUXILIARIES 5.4.2.3 SAMPLE PIPE FOR SCREENS The sample pipe for screens has a very small dead volume. The height-adjustable sample pipe can be used with or without a sample screen. If it is used without sample screen, the small dead volume of the pipe guarantees samples from the culture that are representative for the reactor contents. If a sample screen is used at the end of this pipe, cell-free samples can be drawn from the culture. Sample screens are available in several pore sizes. Z81319MB09 Sample Pipe 2 to 5 liter 10 mm port Z81319MB11 Sample Pipe 7 to 20 liter 10 mm port Z811303010 Sample Screen pore size = 13 μm Z811303011 Sample Screen pore size = 25 μm Z811303012 Sample Screen pore size = 76 μm Z811303013 Sample Screen pore size = 105 μm 5.4.2.4 NARROW SAMPLE PIPE This sample pipe is designed for the sampling of small volumes. The small internal diameter of the pipe guaranties a minimum dead volume. The sample pipe fits into a 6 mm (baffle) port or into a 10 mm port, depending on type number. Diameter ID = 1.56 mm Z81319MB13 Sample Pipe 2 and 3 liter 6 mm port Z81319MB15 Sample Pipe 2 to 7 liter 10 mm port 5.4.2.5 WIDE SAMPLE PIPE This sample pipe is designed for sampling cultures with flocculating organisms. In this case a sample pipe with a small diameter would ruin the flocks and the pipe would be clogged. The shear forces inside this sample pipe are nearly negligible. The sample pipe can be fitted in the ph sensor nipple (ID = 12 mm). Diameter: ID = 10 mm Z81319MB22 Sample Pipe 1 to 5 liter 12 mm ph sensor nipple Z81319MB23 Sample Pipe 7 liter 12 mm ph sensor nipple Z81319MB24 Sample Pipe 15, 20 liter 12 mm ph sensor nipple 34