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NEW BRUNSWICK SCIENTIFIC BioFlo CelliGen 115 Manual To Operations

Benchtop fermentor & bioreactor
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Guide to Operations
®
®
BioFlo
/CelliGen
115
Benchtop Fermentor & Bioreactor
MANUAL No: M1369-0050
Revision B
June 2, 2009
New Brunswick Scientific PO Box 4005 44 Talmadge Rd. Edison, 08818-4005 USA
www.nbsc.com
1.800.631.5417 1.732.287.1200 bioinfo@nbsc.com

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  • Page 1 Guide to Operations ® ® BioFlo /CelliGen Benchtop Fermentor & Bioreactor MANUAL No: M1369-0050 Revision B June 2, 2009 New Brunswick Scientific PO Box 4005 44 Talmadge Rd. Edison, 08818-4005 USA www.nbsc.com 1.800.631.5417 1.732.287.1200 bioinfo@nbsc.com...
  • Page 2 BioFlo/CelliGen 115 • M1369-0050 User’s Guide...
  • Page 3 Fax: 49 (0)7022 32486 E-mail: sales@nbsnv-sa.be E-mail: sales@nbsgmbh.de CHINA THE NETHERLANDS New Brunswick Scientific Co., Inc. New Brunswick Scientific BV A903, 904 Yin Hai Building Kerkenbos 1101, 6546 BC Nijmegen No. 250, Cao Xi Road P.O Box 6826, 6503 GH Nijmegen Shanghai 200235, P.R.
  • Page 4 Do not use this equipment in a hazardous atmosphere or with hazardous materials for which the equipment was not designed. New Brunswick Scientific (NBS) is not responsible for any damage to this equipment that may result from the use of an accessory not manufactured by NBS.
  • Page 5 Reproduction, adaptation, or translation without prior written permission from New Brunswick Scientific is prohibited. Disclaimer Notice New Brunswick Scientific reserves the right to change information in this document without notice. Updates to information in this document reflect our commitment to continuing product development and improvement.
  • Page 6 W A R R A N T Y Every instrument manufactured by New Brunswick Scientific is warranted to be free from defects in material and workmanship. This apparatus with the exception of glassware, lamps and electrodes (where supplied), is warranted for 1 year against faulty...
  • Page 7 On this page, record the information for your fermentor/bioreactor and retain this for future reference. MODEL NUMBER: ________________________________ VOLTAGE: ________________________________ SERIAL NUMBER: ________________________________ The above information can be found on the electrical specification plate. Purchased with the following installed options: __________________________________________________ __________________________________________________ __________________________________________________ __________________________________________________ __________________________________________________ New Brunswick Scientific User’s Guide...

  • Page 8: Table Of Contents

    viii TABLE OF CONTENTS WARNINGS & CAUTIONS ................... 1 WARNINGS ......................1 CAUTIONS........................ 2 INSPECTION & UNPACKING OF EQUIPMENT............. 4 ) ....................4 NSPECTION OF ..................4 ACKING ERIFICATION ....................4 ASIC OMPONENTS INTRODUCTION & OVERVIEW ................5 ........................5 YSTEM ........................
  • Page 9 Cascade Screen ....................58 6.2.11 Pump Screen..................... 59 6.2.12 Setup Screen ..................... 59 PROBE PREPARATION & CALIBRATION............62 ....................62 ROBE NSPECTION ................... 62 ROBE ALIBRATION 7.2.1 pH Probe Installation..................63 7.2.2 pH Probe Maintenance & Storage..............65 New Brunswick Scientific User’s Guide...
  • Page 10 (DO) P ............. 66 ISSOLVED XYGEN ROBE REPARATION 7.3.1 Inspecting the DO Probe.................. 66 7.3.2 DO Probe Preparation..................66 7.3.3 DO Probe Installation..................67 7.3.4 DO Probe Polarization ..................68 7.3.5 DO Probe Calibration: Setting Zero............... 68 7.3.6 DO Probe Calibration: Setting Span .............. 70 ..................
  • Page 11 Motor Assembly Replacement ................ 112 16.6 ..................... 113 EPLACEMENT ARTS SERVICE ........................114 17.1 ....................114 ROUBLESHOOTING DRAWINGS ......................116 18.1 ....................116 IST OF RAWINGS 18.2 ....................... 117 IST OF ABLES APPENDIX A: SOME GENERAL CONCEPTS..........118 New Brunswick Scientific User’s Guide...
  • Page 12 19.1 ?..................118 HAT IS A ONTROLLER 19.2 ? ................... 118 HAT IS A ONTROL 19.3 ? ................119 HAT IS ROBE ALIBRATION 19.4 P-I-D C ?................119 HAT ARE ONSTANTS 19.5 P-I-D T ? ..................120 HAT IS UNING 19.6 ? ................

  • Page 13: Warnings & Cautions

    Always turn your BioFlo/CelliGen 115 off and disconnect the power cord 111, 114 before performing maintenance. NO ONE BUT A PROFESSIONAL SERVICE PERSON should touch electric or electronic parts or assemblies in the electrical cabinet. New Brunswick Scientific User’s Guide...

  • Page 14: Cautions

    CAUTIONS CAUTION Page This equipment must be operated as described in this manual. If operational guidelines are not followed, equipment damage and personal injury can occur. … Do not use this equipment in a hazardous atmosphere or with hazardous materials for which the equipment was not designed. Before making electrical connections, verify that the supply voltage matches the voltage and the power requirements marked on the electrical specification 12, 15...
  • Page 15 Proper pH control is critically dependent on tubing size, which should be as small as possible. Never clean the vessel or its components or the control cabinet with abrasive chemicals or materials. Never let a DO probe rest on its tip. New Brunswick Scientific User’s Guide...

  • Page 16: Inspection & Unpacking Of Equipment

    & Inspection of Box(es) When you have received your order from New Brunswick Scientific, carefully inspect all parts of the shipment for damage that may have occurred during shipping. Report any damage immediately to the carrier and to your local NBS Sales Order Department.

  • Page 17: Introduction & Overview

    DO. (See Section 10 for further information on setting up cascades.) Default P & I (proportional & integral) values are preset at the factory. We strongly recommend that you maintain the factory-set parameters. New Brunswick Scientific User’s Guide...

  • Page 18: Temperature Control

    Temperature Control The culture temperature setpoint may be selected within the range from 20°C above coolant temperature to 70°C for 1.3- to 7.5-liter vessels, and from 20°C above coolant temperature to 65°C for 14.0-liter vessels. It is controlled by the process control software which then sends information to either a heater blanket and cooling coil or to a water jacket.

  • Page 19: Do Control

    NEVER block the exhaust to pressurize the vessel! 3.10 Recommended Accessories & Supplies Before you begin to assemble your BioFlo/CelliGen 115, it would be prudent to verify that you have all of the following accessories and supplies readily at hand: New Brunswick Scientific User’s Guide...

  • Page 20: Supervisory Software

    • An autoclave • An inoculation syringe • Rubber gloves • Media • Silicone tubing • Antifoam agent • A tie gun • Aluminum foil • Plastic ties (multiple colors can be helpful) • Rubber bands • Plastic tubing connectors •...

  • Page 21: Installation

    Figure 1a: Dimensions (viewed from the top) 16.00" Environment The BioFlo/CelliGen 115 fermentor operates properly under the following conditions: • Ambient temperature range 10°C to 35°C • Relative humidity up to 80% non-condensing New Brunswick Scientific User’s Guide...

  • Page 22: Installing The Control Cabinet

    Installing the Control Cabinet Position the BioFlo/CelliGen 115 control station cabinet on a firm, level surface in an area where utilities are readily available. Connect the power cord to the rear of the control cabinet. At a later time, once the unit is completely assembled and all connections have been made, you will plug the power cord into a suitable electrical outlet.
  • Page 23 (see Section 4.5.3) Service Connections (see Figure 1d) Cabinet Input port (see Section 4.4) Cabinet Output port (see Section 4.4) Label with electrical specifications & unit serial SCADA port number (see Section 4.10) Plug for Power Cord New Brunswick Scientific User’s Guide...

  • Page 24: Connecting Utility Cabinets

    Figure 1d: Control Station Service Connections Touchscreen, gas and pump control options may or may not be present, depending on the configuration of your control station. These connections are addressed in Section 4.5.2. CAUTION! Before making electrical connections, verify that the supply voltage matches the voltage and the power requirements marked on the electrical specification plate (located on the rear panel of the cabinet) and the control schematics supplied with the unit.
  • Page 25 1st unit..to INPUT of 2nd unit. To add a second utility station, repeat Steps 1 & 2. Section 12.3 explains how to set up the control station and the utility station(s) to work together. New Brunswick Scientific User’s Guide...

  • Page 26: Utilities

    Utilities WARNING! Do not use this equipment in a hazardous atmosphere or with hazardous materials for which the equipment was not designed. All control and utility stations must be properly connected to gases, water supply, vessel water, electrical power and an open drain. The gas connections are located on the rear panel of the cabinet (see Figure 1g in Section 4.5.3).

  • Page 27: Water And Drain Connections

    BE SURE TO READ THE (2) to the water inlet and outlet lines coming from water jacketed vessels. The connection points on ON THE NEXT PAGE. CAUTION the open ends should be secured with cable ties. New Brunswick Scientific User’s Guide...

  • Page 28: Gas Connections

    CAUTION! Before connecting or disconnecting the water hoses to/from the vessel and/or the cabinet at any time, make sure the main water supply is closed. 4.5.3 Gas Connections Gas inlets are located on the rear panel of the control cabinet (see Figure 1g on the following page).

  • Page 29: Important Warnings

    **Important Warnings** Before you begin to assemble or operate your vessel, be sure to read this section, for it contains essential information, cautions and warnings to protect your safety and the safety of your equipment. New Brunswick Scientific User’s Guide...
  • Page 30 WARNING! NEVER PRESSURIZE A GLASS VESSEL! • Always use eye protection, and exercise caution in the vicinity of glass. If the vessel exhaust becomes blocked, pressure can build up, possible shattering the vessel and endangering personnel. • As soon as you open the airflow valve(s), verify by feel that air is flowing freely from the exhaust.

  • Page 31: Vessel Assembly: Non-Jacketed

    Figure 2b: CORRECT Handling of Drive Assembly HEADPLATE DRIVE ASSEMBLY IMPELLER SHAFT Vessel Assembly: Non-Jacketed The vessels are available in four sizes: 1.3 liters, 3.0 liters, 7.5 liters and 14.0 liters (total volume; for more detail, see Specifications). New Brunswick Scientific User’s Guide...
  • Page 32 Every single-walled, non-jacketed vessel comes with a stainless steel stand from which the vessel is suspended. The stand has four rubber feet to provide stability. An electric heat blanket provides temperature control for the contents of the vessel. The blanket (shown in the smaller vessel views in Figure 3 on the following page) has two large viewing windows so the culture remains visible for inspection.
  • Page 33 Familiarize yourself with the arrangement of the headplate ports, as shown in the following diagrams, before proceeding with the vessel assembly. You may find it more practical to change the arrangement; the variety of ports and adapters will easily accommodate your needs. New Brunswick Scientific User’s Guide...

  • Page 34: Headplate

    4.7.1 Headplate Figure 4: 1.3L Headplate EXHAUST SPARGER/ COOLING COIL dO2 PROBE HARVEST/SAMPLER/SPARE ASSEMBLY RTD THERMOWELL (see NOTE below) Each bolt is a possible mounting position for a bottle holder NOTE: The RTD thermowell port should only be used for its intended purpose. BioFlo/CelliGen 115 M1369-0050 User’s Guide...
  • Page 35 EXHAUST COOLING COIL Mounting position for dO2 PROBE Sampler Assembly. RTD/THERMOWELL Each bolt is a possible mounting position for a bottle holder. NOTE: The RTD thermowell port should only be used for its intended purpose. New Brunswick Scientific User’s Guide...

  • Page 36: Install Heat Blanket

    Figure 6: 7.5L & 14.0L Headplate COOLING COIL COOLING COIL EXHAUST dO2 PROBE LIFTING HANDLE LIFTING HANDLE THERMOWELL SEPTUM PLUG 19mm/SPARE Each bolt is a possible mounting position for a bottle holder. NOTE: The RTD thermowell port should only be used for its intended purpose. 4.7.2 Install Heat Blanket 1.

  • Page 37: Install Vessel In Vessel Stand

    4. Orient the vessel so the gradations on the glass are clearly visible at the front, facing the user, and situated between two vessel stand pillars. New Brunswick Scientific User’s Guide...

  • Page 38: Install Baffle (14.0L Fermentation Vessels Only)

    4.7.4 Install Baffle (14.0L Fermentation Vessels ONLY) For installation of the 1.3L, 3.0L and 7.5L vessel baffle, see Section 4.8.21. If you are using a 14.0L vessel, install the baffle assembly inside the glass vessel: 1. Gently compress the baffle ring at its ends (to avoid scratching the vessel walls).
  • Page 39 LIFTING HANDLE HEADPLATE TOP CLAMPING RING BAFFLE COOLING WATER OUTLET TUBE WATER JACKET SPARGER BOTTOM CLAMPING THERMOWELL RING BASE PLATE COOLING WATER OUTLET (COOL COOLING WATER INLET (COOL LOOP RETURN—tubing connected LOOP IN) inside the jacket) New Brunswick Scientific User’s Guide...

  • Page 40: Install Headplate Clamping Ring

    4.8.1 Install Headplate Clamping Ring The clamping ring that secures the headplate to the vessel is split in half to facilitate installation under the vessel flange. They are joined with two rectangular mounting plates. 1. As shown in Figure 9 below, install one mounting plate with two Phillips head screws (provided) on the end of one ring half so that the plate extends beyond the ring.

  • Page 41: Filling The Water Jacket

    4.8.4 Install Baffle (14.0L Fermentation Vessels ONLY) For installation of the 1.3L, 3.0L & 7.5L vessel baffle, see Section 4.8.21. If you are using a 14.0L vessel, install the baffle assembly inside the glass vessel: New Brunswick Scientific User’s Guide...

  • Page 42: Install Impeller(S)

    1. Gently compress the baffle ring at its ends (to avoid scratching the vessel walls). You may find it convenient to squeeze the tab with your thumb. 2. Slide the assembly inside, with the tab facing up, until it comes to rest at the bottom of the vessel.

  • Page 43: Install Cooling Coil

    Do not overtighten. CAUTION! Finger tighten only any adapter that has a white Teflon ferrule (tapered, cone-shaped insert under the Teflon washer). The ferrule can deform under too much pressure. New Brunswick Scientific User’s Guide...

  • Page 44: Install Harvest Tube

    4.8.8 Install Harvest Tube 1. Working from beneath the headplate, install the harvest tube in the harvest port. If you are using the 1.3L vessel, the harvest tube and sampler tube are welded into the same tri-port to save space. When the headplate is in place on the vessel, the bottom of the harvest tube should rest at the bottom of the vessel.

  • Page 45: Install Foam Exhaust Tube

    Prior to installation, any pH probe you are using should be inspected for damage, and replaced if necessary. NOTE: To avoid damage to the probes during operation, be sure that there is no interference between the probes and the baffle assembly, impeller blades, or cooling coil. New Brunswick Scientific User’s Guide...
  • Page 46 1. Wear protective gloves to protect yourself in case of accidental breakage. 2. Lightly coat the pH probe with glycerol. CAUTION! Do not install the pH port adaptor in the headplate before inserting the probe. Follow the steps below to fit the pH port adapter onto the probe first, then insert the probe and adapter into the headplate.

  • Page 47: Install Do2 Probe

    Do not install the dO2 port adaptor in the headplate before inserting the probe. Follow the steps below to fit the dO2 port adapter onto the probe first, then insert the probe and adapter into the headplate. New Brunswick Scientific User’s Guide...
  • Page 48 Figure 12: dO2 Probe with Port Adapter (exploded) dO2 PROBE ADAPTER (top portion) TOP FERRULE BOTTOM FERRULE dO2 PROBE ADAPTER (bottom portion) TEFLON O-RING (white) PORT O-RING (black) With reference to Figure 12 above: 3. Gently slide the top portion of the knurled port adapter (part of the probe kit) onto the probe.

  • Page 49: Install Exhaust Condenser

    If the weight of the exhaust filter kinks the tubing, fasten a short length of stiffening material to the tubing, using rubber bands or tie wraps, to support the filter. Ensure that gas flow through the exhaust condenser is unobstructed during runs and during autoclaving. New Brunswick Scientific User’s Guide...

  • Page 50: Install Sampler

    Figure 13b: Exhaust Condenser (14.0L Vessel Only) STERILE FILTER WATER OUTLET (EXH. COND. RETURN) WATER INLET (EXH. COND. IN) SET SCREW in port adapter HEADPLATE EXHAUST PORT 4.8.18 Install Sampler The optional BioFlo/CelliGen 115 sampler system is designed to aseptically remove batch samples from the vessel.
  • Page 51 HARVEST TUBE Figure 14b: Sampler System (3.0L, 7.5L & 14.0L Vessels) 3/16” SILICONE TUBING THUMB CLAMP SAMPLER PORT SYRINGE HEADPLATE SYRINGE FILTER SAMPLER BOTTLE HOLDER SAMPLER BOTTLE HEADPLATE CLAMPING NUT SAMPLER TUBE HEADPLATE CLAMPING SCREW New Brunswick Scientific User’s Guide...

  • Page 52: Install Foam Trap

    3. Connect a length of silicone tubing to the sampler tube on the headplate. Secure it in place with a plastic tie. 4. Slip a thumb clamp onto the tubing. 5. Connect the other end of the tubing to the tall sampler inlet pipe. Secure it in place with a plastic tie.

  • Page 53: Plug Unused Ports

    1. Install a blind plug (without a hole) in any headplate port that will not be used. 2. Install silicone tubing, secured with a plastic tie and clamped shut, on any access tube (i.e., harvest tube) that will not immediately be used. New Brunswick Scientific User’s Guide...

  • Page 54: Install 1.3L, 3.0L Or 7.5L Fermentation Vessel Baffle

    NOTE: It is good practice to lightly lubricate the underside of the headplate with silicone before installing it on the vessel. 4.8.21 Install 1.3L, 3.0L or 7.5L Fermentation Vessel Baffle 1. Gently place the baffle, tab facing up, around all of the other instruments protruding from the headplate, including the cooling coil.

  • Page 55: Install Vessel

    2. Connect the motor cable to the receptacle on the face of the control cabinet. 4.8.25 Make All Connections 1. Connect cables from all probes to their respective sockets on the face of the control cabinet (see Figure 1d). New Brunswick Scientific User’s Guide...

  • Page 56: Main Power Switch

    2. Connect the exhaust condenser to the exhaust condenser port. 3. Using flexible tubing, connect the exhaust filter to the top of the condenser. Secure it with tubing ties. 4. Insert the RTD into the thermowell. 5. If you have not already done so, connect the sparge line (silicone tubing) to the inlet filter.

  • Page 57: Optional Biocommand Software

    A 25-pin RS232/422 “D” connector com port, labeled , is provided on the rear SCADA panel of the control cabinet (see Figure 17a) to connect the BioFlo/CelliGen 115 to a supervisory host computer. Figure 17a: RS232/422 Interface SCADA (RS232/422) connector New Brunswick Scientific User’s Guide...
  • Page 58 Communication to BioCommand software is assured via an optional RS-232 interface cable: 1. Connect the 25-pin end of the RS-232 cable to the port, and make sure that SCADA the connection is secure. 2. Hand tighten the thumbscrews. 3. Refer to the BioCommand user’s guide for instructions on connecting the RS-232 interface cable to the supervisory host computer.

  • Page 59: Specifications

    Optional: Microsparger 0.2μm interchangeable cartridge Inlet Filter For calibration of DO probe Digital display in 0.01 pH increments Indication 2-14 pH Range PID, link to pumps or gases, adjustable deadband Control pH probe Sensor ...continued... New Brunswick Scientific User’s Guide...

  • Page 60: Certifications

    BioFlo/CelliGen 115 System Digital display in 0.1% increments Indication 0-200% Range PID, Cascade to Agitation, Gases, GasFlo if equipped with TMFC Control Polargraphic DO probe Sensor 0.2μm interchangeable cartridge Exhaust Filter Stainless steel, water-cooled in headplate Condenser 12 RPM 3 Pumps Control 10 PSIG maximum, 50 μm filtration Utilities...
  • Page 61 New Brunswick Scientific User’s Guide...

  • Page 62: Operating Controls

    Touchscreen Your primary interface with the BioFlo/CelliGen 115 is the touchscreen on the control cabinet. Figure 18: Touchscreen CONTROL TOUCHSCREEN CABINET DISPLAY: Only the control station is equipped with a touchscreen; utility stations do not have one. ROTAMETERS: You may have from 0 to 4 rotameters.

  • Page 63: Start-Up Screen

    The available loops will change depending on your system’s configuration. This one is equipped with automatic Gas Mix. SCREEN ACCESS buttons NOTE: The dark blue button usually represents the screen being displayed. New Brunswick Scientific User’s Guide...
  • Page 64 Figure 19b: Sample SUMMARY Screen (Fermentation with Manual Gas Mix) OPERATING SCREEN UNIT TABS MODE NAME and ICON Current DATE & TIME Your BioFlo/CelliGen 115 comes with pre-assigned loop names. The available loops will change depending on your system’s configuration. This one is equipped with manual Gas Mix.
  • Page 65 Depending on the loop, the control mode may be Off, Auto, Manual, On, or O2 Enrich. Unit (of measure) This is the unit of measure used for the PV and Setpoint. Cascade If any cascades have been programmed, they will be displayed here. …continued… New Brunswick Scientific User’s Guide...

  • Page 66: Keypads

    Navigation Buttons Description (for screen access) Summary This screen is command central; it shows all your loops, their current readings, setpoints and what has been programmed for them. Calibration This screen allows you to calibrate the pH, DO & Level probes and the gas flow.

  • Page 67: Gauge Screens

    (See Section 19.4 for to reflect the high & Decimal Places: more information on P&I low limits selected. Gains.) Press the appropriate button to display values with 0, 1, 2 or 3 decimal places. New Brunswick Scientific User’s Guide...

  • Page 68: Selecting Loop Control Modes

    6.2.6 Selecting Loop Control Modes A control mode is the logic by which a controller generates the desired control signal. The operator has a choice of control modes, the most common of which are AUTO MANUAL . Other available control modes, in certain cases, are O2ENRICH 2-GAS...

  • Page 69: Modifying Setpoints

    This process is the same as entering setpoints. See Section 6.2.7 above. NOTE: Sections 6.2.9 - 6.2.12 will acquaint you with the primary screens accessed from the blue buttons at the bottom of each screen. New Brunswick Scientific User’s Guide...

  • Page 70: Calibration Screen

    6.2.9 Calibration Screen This screen is used to calibrate the pH, DO and level probes. For details on probe calibration, see Sections 7.2 (pH probe), 7.3 (DO probe) and 7.4 (Level probes). Figure 23: Calibration Screen These last two “loops” are input from the Level probes to the Level1 and Level2 loops.

  • Page 71: Pump Screen

    For details on using the SETUP screen, see Section 12. When you press the SETUP button, the screen that opens is actually the first CONTROLLER SETUP tab, the (see Figure 26) screen: New Brunswick Scientific User’s Guide...
  • Page 72 Figure 26: Controller Setup Screen The UNIT NAME is user-selected. Press this box, then use The TMFC Range the pop-up keypad and the number of to type in the name. TMFCs (0 means manual gas flow, The VESSEL SIZE is usually by user-selected by rotameter) are...
  • Page 73 3 units installed in the system, and to set Unit IDs for software (see Section 12.3 ). Use this pane to choose software connections (as explained in Section 12.3 ). New Brunswick Scientific User’s Guide...

  • Page 74: Probe Preparation & Calibration

    & & pH Probe Inspection Inspect probe for possible shipping damage. If damage is observed, notify the New Brunswick Scientific Service Department immediately. Check the electrode tip for trapped air bubbles. To remove any air bubbles, hold the electrode upright and shake gently. NEVER REST THE PROBE ON ITS TIP. pH Probe Calibration NOTE: Calibrate the pH probe before autoclaving it with the vessel.

  • Page 75: Ph Probe Installation

    SET ZERO pH meter. Any discrepancy should be adjusted with the procedure. 7.2.1 pH Probe Installation CAUTION! Be sure to wear protective gloves when installing a glass electrode. New Brunswick Scientific User’s Guide...
  • Page 76 NOTE: Prior to installation, any pH probe you are using should be inspected for damage, and replaced if necessary. NOTE: To avoid damage to the probes during operation, be sure that there is no interference between the probes and the baffle assembly, impeller blades, or cooling coil.

  • Page 77: Ph Probe Maintenance & Storage

    4.00 and pH 7.00. If the probe is so equipped, the two rubber T stoppers should be inserted. CAUTION! Never let a pH probe rest on its tip, and never leave a pH probe in DI water. New Brunswick Scientific User’s Guide...

  • Page 78: Dissolved Oxygen (Do) Probe Preparation

    7.3.1 Inspecting the DO Probe Inspect the probe for possible shipping damage. Immediately report any damage you may observe to the New Brunswick Scientific Service Department. Remove the protective cap from the electrode end. The membrane is delicate and care must be exercised to prevent accidental damage. NEVER REST THE PROBE ON ITS MEMBRANE.

  • Page 79: Do Probe Installation

    PROBE ADAPTER (bottom portion) TEFLON O-RING (white) PORT O-RING (black) With reference to Figure 29 above: 3. Gently slide the top portion of the knurled port adapter (part of the probe kit) onto the probe. New Brunswick Scientific User’s Guide...

  • Page 80: Do Probe Polarization

    4. Slide the two white ferrules onto the probe, the narrower one on top of the deeper, cup-shaped one. 5. Gently slide the bottom portion of the port adapter onto the probe, taking care to orient the longer threaded section toward the top of the probe. 6.
  • Page 81 DO value by the controller. As explained in Step 4 above, these buttons may not be present for your system’s configuration. New Brunswick Scientific User’s Guide...

  • Page 82: Do Probe Calibration: Setting Span

    7.3.6 DO Probe Calibration: Setting Span 1. In the AGIT GAUGE screen, set the AGIT speed to 50 RPM. 2. Set the AGIT mode to AUTO 3. Vigorously sparge air into the vessel via the filter on the headplate until the display is stable for approximately 10 minutes (this may take up to 30 minutes total).

  • Page 83: About Pump Calibration

    About Pump Calibration To assure the most accurate flow rate, calibrate the pump each time you change tubing. See Section 11.6.4 for details. New Brunswick Scientific User’s Guide...

  • Page 84: Vessel Sterilization

    NOTE: Before proceeding, consult the dimensions of your vessel assemblies to be sure your autoclave is large enough to accommodate the vessel with its various components. WARNING! During autoclaving, the vessel exhaust filter must be vented to avoid explosion. WARNING! Use protective gloves when handling hot components.

  • Page 85: Initial Preparation For Autoclaving

    14. Reinsert the bottle and turn the holder until the bottle and holder are positioned over the headplate, rather than extended over the edge. 15. Finger tighten the knurled nut. 16. Clamp off the tubing, and, where appropriate, remove it from the pump. New Brunswick Scientific User’s Guide...

  • Page 86: Autoclaving The Vessel

    Probe tips must be moist during sterilization: • If you will be doing batch fermentation, be sure the vessel is filled with media so the media will also be sterilized. • If you will be using heat-liable media, use at least 100 ml of a balanced salt solution (such as phosphate-balanced saline solution).

  • Page 87: Sterilization Time And Temperature

    Adjust the time and temperature as needed. If, after autoclaving, most of the liquid has left the vessel, the autoclave is exhausting too quickly. Adjust the autoclave to exhaust more slowly. New Brunswick Scientific User’s Guide...

  • Page 88: Reinstalling The Vessel Assembly

    Reinstall the Vessel Assembly WARNING! Cold water and hot glass is a potentially dangerous mix! Be sure to let the vessel cool for a few minutes before reconnecting the water line. 1. Position the vessel next to the BioFlo/CelliGen 115 control cabinet. Connect the water lines to the heat exchanger and the exhaust condenser (see Vessel Assembly section).
  • Page 89 2. Select the desired tubing size (see Table 6 in Section 11.4 for reference) and cut a length sufficient to reach from the inlet source, through the pump, and to the outlet recipient, allowing a few extra inches. 3. Form a loop large enough to go around the pump head. New Brunswick Scientific User’s Guide...

  • Page 90: Confirm Ph Calibration

    4. Fit one side of the tubing loop into one of the spring-loaded clamps, pulling the clamp open with your finger. WARNING! Be careful not to pinch your fingers in the pump head levers. 5. Then, as you rotate the pump’s rotor by hand in a clockwise direction to clear the channel, lay the tubing in the channel around the pump head.

  • Page 91: Addition Tubing Size

    Figure 34: Typical Liquid Addition System TUBING PERISTALTIC PUMP BREATHING ADDITION PORT with PORT PLASTIC STERILE TIES FILTER (0.2 µ) ADDITION BOTTLE New Brunswick Scientific User’s Guide...

  • Page 92: Reconnect Gases

    The concentrations of acid and base, and the inner diameter of the acid and base addition tubing (where they pass through the peristaltic pumps), are critical parameters in the proper operation of a P&I pH control system. If the tubing is too large, excessive doses will be added. The result is that the system will “overcontrol,”...

  • Page 93: Cascade Control

    DO still has not reached the setpoint, increasing the GasFlo entering through the sparger to as much as the high limit. This cascade is most frequently used in fermentation. (This is not available without the Automatic GasFlo Option.) New Brunswick Scientific User’s Guide...

  • Page 94: Creating A Cascade

    • GasFlo/O2 controls dissolved oxygen by first increasing GasFlo to the high limit, then, if DO still has not reached the setpoint, increasing oxygen percentage entering the system through the sparger to as much as the high limit. (This is not available without the Automatic GasFlo Option and Automatic Gas Mix Option.) •...
  • Page 95 In this sample cascade, as the system demands an increase of DO, agitation will increase from 25 to 200. If there is still a need for more DO, the GasFlo loop will kick in until the need is satisfied. New Brunswick Scientific User’s Guide...

  • Page 96: About Pumps

    After assigning the pumps (see Section 11.1), you will need to select a setpoint and a control mode for each, calibrate their flow rates, and select their pulse periods. This section will walk you through those operations. There are three standard pumps on the front right of your control cabinet (see Figure 32, repeated here for easy reference).

  • Page 97: Pump Setpoint

    For details on the choice of Level Wet and Level Dry, see Section 11.6.1. 11.2 Pump Setpoint To enter a setpoint for a pump: 1. Open the PUMP screen. Gauges for Pumps 1-3 are displayed in this screen (see Figure 36 on the following page). New Brunswick Scientific User’s Guide...
  • Page 98 Figure 36: Setting Pump Setpoint 2. Press the Setpoint edit box for Pump. 3. Use the touchpad that opens to enter the desired setpoint, then press OK to save it and return to this screen (or press Cancel to return to this screen without saving a setpoint.

  • Page 99: Pump Control Mode

    DO NOT allow the liquid to run into the graduated cylinder yet. New Brunswick Scientific User’s Guide...

  • Page 100: Pump Period

    4. If you are not using a scale, skip to Step 5. If you are using a scale, place the graduated cylinder (with the tubing) on the scale and press Zero on the scale. 5. In the Flow Rate pane of the PUMP screen (see Figure 36) for that pump, press the Calibrate...

  • Page 101: Using Level Probes To Program Feed Pumps

    • when the liquid is not in contact with the probe, the feed pump is turned on so that more liquid will be added. • when the liquid is in contact with the probe, the pump is turned off. New Brunswick Scientific User’s Guide...

  • Page 102: Setting A Feed Pump To Harvest

    11.6.2 Setting a Feed Pump to Harvest A level probe can also be used to set up a feed pump to harvest. Prior to autoclaving the vessel make sure that the level probe that you wish to use is fully inserted into the vessel. When the vessel is set up at the control cabinet, raise the probe to the level at which you want harvesting to begin (i.e., above the current liquid level).
  • Page 103 NOTE: To assure the most accurate flow rate, calibrate the pump (see Section 11.4) each time you change tubing. New Brunswick Scientific User’s Guide...

  • Page 104: Using The Setup Screen

    SETUP screen (see Figure 39) is used to change Controller Setup (see Section 12.1), to adjust System Settings (select onscreen language when available, change date & time, update software and calibrate the touchscreen; see Section 12.2), and to check or change the Hardware Setup (see Section 12.3).
  • Page 105 The mode will also change in the upper righthand corner of the SETUP screen when you press the Save Changes button (see Figure 40a). Figure 40a: Operating Control Mode Changed The new Operating Control Mode also appears here. New Brunswick Scientific User’s Guide...

  • Page 106: Gas Control

    If you run the unit with various vessel sizes or the size indicated is incorrect for the Unit indicated, use the Vessel Size dropdown menus to change to the new vessel size (see Figure 40b), then press the Save Changes button to allow the system to reset to new parameters.
  • Page 107 4 Gas gives you a 4- Gas button, as shown in Figure 41b. Selecting Manual in any other gas gauge screen allows you to adjust the percentage of that gas; air always makes up the remainder (if any) of 100%. New Brunswick Scientific User’s Guide...
  • Page 108 When you have Cell Culture as the Control Mode and 4-Gas mix as the Operating Mode, the process loops are labeled Air (1)—as shown in Figure 41b—O2 (2), N2 (3) and CO2 (4). The loops’ numbers, 1, 2, 3 & 4, correspond to the gas connections on the cabinet (see Figure 1g).

  • Page 109: System Settings

    12.2.2 Updating Software To update the system software, obtain a new version of the software in a USB drive and plug the drive into the USB port on the control cabinet: New Brunswick Scientific User’s Guide...

  • Page 110: Hardware Setup

    1. In the System Settings screen, press the Refresh button to update the current software status and to search for a new USB drive. 2. The name of the new drive folder appears in the Update File box. 3. Press the Update button to install the file.
  • Page 111 Figure 45a: Adding New Hardware When you press the Scan Hardware button, new hardware appears here. Figure 45b: New Hardware Added When you press the >>> button for the Unit involved, the new hardware moves into this pane. New Brunswick Scientific User’s Guide...

  • Page 112: Identifying Utility Station(S) Added

    12.3.1 Identifying Utility Station(s) Added Now that you have added one or more utility station(s) using the Hardware Setup Controller Setup screen, return to the screen to name the new unit(s) as desired, and to identify the vessel size, the operating mode and the options installed on the unit.

  • Page 113: Performing A Run

    Preparing for a Fermentation Run 1. Connect water to the unit and turn it on. 2. Make sure the drain line is properly connected to the unit. 3. Connect the quick-connect plastic water lines to the exhaust condenser. New Brunswick Scientific User’s Guide...

  • Page 114: Inoculation

    CAUTION! Before connecting or disconnecting the water hoses to/from the vessel and/or cabinet at any time, make sure the main water supply is closed. 4. Add glycerin to the thermowell and insert the temperature probe. 5. Make sure the motor is not connected. Turn the power 6.

  • Page 115: Start Biocommand (If Present)

    Lag phase, (2) the Exponential Growth phase, (3) the Steady State phase, and (4) the Decline phase. 13.6.1 Lag Phase This initial phase is aptly named because it is the slow beginning of your fermentation run, while the microbes become accustomed to their medium. New Brunswick Scientific User’s Guide...

  • Page 116: Exponential Growth Phase

    13.6.2 Exponential Growth Phase After the initial lag, a sudden spurt in growth will indicate that the environment is fully hospitable to the microbes. Compared to the nearly inanimate lag phase, this activity will appear to be nearly uncontrolled. 13.6.3 Steady State Phase Most of your run will be the desired steady state of growth.

  • Page 117: Continuous Operation

    BROTH MEDIA 13.10 Anaerobic and Microaerophilic Culture When growing anaerobic organisims, oxygen must be excluded from the media, and when growing microaerophilic organisms, oxygen must be limited to a very low level in the media. New Brunswick Scientific User’s Guide...

  • Page 118: Harvesting Procedure

    For anaerobes, several strategies can be used to eliminate oxygen: • Reducing agents can be added to the media. • Vigorous agitation (normally used to increase dissolved oxygen in the media) is not required. A low agitation rate, however, is required to keep the cells in suspension and to provide mixing of the liquid to maintain good temperature control.
  • Page 119 6. Remove, drain and clean the vessel as outlined in Section 15. See also Section 21.7.5 for shutdown and cleaning tips. NOTE: Never wash the filters or get them wet. New Brunswick Scientific User’s Guide...

  • Page 120: Essential Operating Tips

    14.1 Precautions for Glass Vessel Assembly There are certain precautions you should take to avoid cracking or breaking the glass vessel during assembly and autoclaving: • Glass can crack or break during assembly if the clamping screws are overtightened. As a precaution, tighten the screws only finger tight prior to autoclaving.

  • Page 121: Install A Double Filter System

    2. Attach an exhaust filter to each branch of the Y. This allows you the flexibility to exchange sterilized filters during a run should one filter become clogged: all you have to do is pinch off the unused line with a clamp. New Brunswick Scientific User’s Guide...

  • Page 122: Cleaning

    CAUTION! Never clean the vessel or its components or the control cabinet with abrasive chemicals or materials. 15.1 Cleaning the Vessel NOTE If applicable, be sure to follow the bio-safety regulations regarding the release of microorganisms into the environment. 1. Fill the vessel with a mild detergent and water solution. 2.

  • Page 123: Maintenance

    At no time should the probe be allowed to rest on its membrane. CAUTION! Never let a DO probe rest on its tip. New Brunswick Scientific User’s Guide...

  • Page 124: Vessel & Tubing

    16.3 Vessel & Tubing After each and every run, clean the vessel and the headplate with its associated parts. All tubing and filters should be replaced. 16.4 Periodic Inspection At three-month intervals, perform the following checks and inspections. NOTE: Before you begin, make sure that the power switch is in the OFF position and that the power supply has been disconnected.

  • Page 125: Replacement Parts

    7.5L, 14.0L M1369-3125 Magnetic Drive All Sizes M1369-3130 Impellers 6-Blade Rushton type 52mm 1.3L, 3.0L M1273-9291 6-Blade Rushton type 59mm 7.5L M1273-9292 6-Blade Rushton type 74mm 14.0L M1273-9293 1.3L, 3.0L M1273-9206 Pitched Blade 7.5l, 14.0L M1273-9207 New Brunswick Scientific User’s Guide...

  • Page 126: Service

    If any problems occur with your BioFlo/CelliGen 115 system or its individual components, do not attempt to perform any service on it. Unauthorized servicing may void the warranty. Please contact your local NBS Service Department or your local NBS distributor. In any correspondence with NBS, please refer to the Model Number (BioFlo/CelliGen 115), and the Manufacturing Part Number and Serial Number of the unit.
  • Page 127 Make sure that the GasFlo loop is ON. • Make sure that the Air loop is in O2 Enrichment mode. • Make sure that the DO cascades are Enabled. GENERAL: Touchscreen is not responding. • Calibrate touchscreen. New Brunswick Scientific User’s Guide...

  • Page 128: Drawings

    18.1 List of Drawings Figure Description Page Dimensions Front View Rear View Control Station Service Connections Connecting Cabinets Water Connections Gas Connections Sparge Connection (detail from Figure 1d) WRONG Handling of Drive Assembly CORRECT Handling of Drive Assembly Vessel Assembly 1.3L Headplate 3.0L Headplate 7.5L &...

  • Page 129: List Of Tables

    Controller Settings for New Hardware Added Fed Batch Operation Continuous Operation 18.2 List of Tables Table Description Page Service Connections Impeller Positions Modbus Com Port Pin Designation SUMMARY Screen Features Pump Control Modes Flow Rate per Tubing Size Wetted Parts New Brunswick Scientific User’s Guide...

  • Page 130: Appendix A: Some General Concepts

    NOTE: In this section, all discussions of P-I-D control are to explain the theory on which it is based. This product uses only P (proportional) & I (integral) control, not D (derivative). 19.1 What is a Controller? The local process controller is a multi-loop controller, which means it can control several process parameters simultaneously.

  • Page 131: What Is Probe Calibration

    If the constants are too small, control response will be slow, and setpoints may never be reached. Again, this can be mistaken for defective components. P-I-D constants are usually established by methodical trial and error. New Brunswick Scientific User’s Guide...

  • Page 132: What Is P-I-D Tuning

    19.5 What is P-I-D Tuning? Tuning consists of establishing controller settings (the proportional, integral, and derivative constants) such that the controller provides proper control. If the P-I-D constants are incorrect, the control signal may be too weak for the parameter to ever reach setpoint or, at the other extreme, the controller may respond excessively to small errors, causing the actuator to oscillate between high and low values.

  • Page 133: What Do The Constants Mean

    0 to 125, setting k to 125 results in a P term value of P when the error is at a maximum, i.e.: /k) = P(125/125) = P Similarly, with a full-scale error, the I term (after 1 minute) and the D term will be I and D respectively. New Brunswick Scientific User’s Guide...

  • Page 134: Appendix B: Otr

    20.1 Determining an Oxygen Transfer Rate The oxygen transfer rate (OTR) of all NBS fermentors is determined by a standard sulfite oxidation test. The standard operating conditions for determining OTR are: Temperature: 30°C Agitation: 1000 RPM Aeration: 1 VVM 20.1.1 OTR Calculations OTR can be estimated by titrating a fixed amount of sodium sulfite, Na with air, CU+2:...

  • Page 135: Some Factors That Affect Otr And Horsepower

    = Constant (factor based on unaerated water at 20°C with a six-bladed Rushton impeller) = factor based on the number of impellers used in the vessel: • Use 1 for one impeller • Use 1.8 for two impellers New Brunswick Scientific User’s Guide...
  • Page 136 • Use 2.4 for three impellers NOTE: The HP requirements are substantially affected by aeration. An airflow rate of one vessel volume per minute (VVM) may produce as much as 40% reduction in the horsepower used. It is required that some air/gas flow be utilized when running at speeds above 750 RPM.

  • Page 137: Appendix C: Fermentation Techniques

    Here is an example of a more complex media used in a recombinant E. coli fermentation: Chemical MgSO Glucose 5.0 (for fed batch) 30.0 (for batch) Yeast Extract Trace Metals 1.0 ml/L Antifoam 0.5 ml/L Trace metals formulation: FeCl CoCl CuCl ZnCl NaMoO 0.05 10 ml to 1000 ml New Brunswick Scientific User’s Guide...

  • Page 138: Antifoam Formulation

    For fermentation, the glucose solution is usually sterilized in a separate flask. It is then added aseptically to the other (heat labile) components that cannot be subjected to autoclaving, such as Ampicillin and the trace metal solution. These are prepared in advance by sterile filtration so that they are available as stock solutions.

  • Page 139: Acid & Base

    This operation can be ® controlled automatically by BioCommand, NBS' proprietary Windows -based software. New Brunswick Scientific User’s Guide...

  • Page 140: Recommended Process Control Settings

    Glucose feeding can be set to respond to other sensor cues (such as DO level, the pH reading, the turbidity measurement, the glucose measurement, etc.). The pumping profile to be used must generally be determined through experimental experience. 21.6 Recommended Process Control Settings Question: What are the recommended process control settings (i.e., temperature, pH, agitation speed, DO &...
  • Page 141 All O-rings should be checked for damage prior to autoclaving. All fittings must be checked for tightness. A loose fitting is often an indication that the small O-ring in the fitting assembly requires replacement. New Brunswick Scientific User’s Guide...

  • Page 142: Vessel Sterilization

    Verify that the bottom of the glass cylinder is properly secured to its base. The agitation shaft must have its protective cap on prior to autoclaving. It is advisable to check that the connectors from the unit to the vessel (exhaust gas condenser) are compatible.

  • Page 143: Post-Sterilization Vessel Set-Up

    GasFlo is in mode. Connect the air line from the unit to the sparger’s terminal filter as aseptically as possible (although the filter will prevent external contamination, good technique is always a good idea). New Brunswick Scientific User’s Guide...

  • Page 144: Vessel Operation

    Open the clamp on the sparger line and visually observe the vessel to ensure that air is flowing properly. Then set the agitation to the minimum desired value. After set-up, the unit should be carefully observed to ensure that there are no problems, (especially no water line leaks).
  • Page 145 Kimwipe should be used to gently dry it off after washing. The probe should be stored with the tip immersed in either electrolyte or pH 7 buffer. This electrolyte/buffer can be reused, but it should always be inspected prior to each use for precipitation or contamination. New Brunswick Scientific User’s Guide...
  • Page 146 Now that the vessel is detached from the unit, it can be cleaned. Remove any remaining cotton and foil covering the ports. The rubber sampler bulb should be removed and rinsed separately. The glass wool can be removed at this point, too.
  • Page 147 In cases where the vessel must be decontaminated prior to cleaning, add water so that the liquid level reaches the maximum working volume of the vessel. This will help prevent biological materials from adhering. New Brunswick Scientific User’s Guide...

  • Page 148: Appendix D: Corrosion Resistance

    Websites such as www.outokumpu.com provide up-to-date information about the 316 type stainless steel used in your BioFlo/CelliGen 115 vessels. BioFlo/CelliGen 115 M1369-0050 User’s Guide...

  • Page 149: Appendix E: General Characteristics Of Epr

    350 (177°C). Ethylene Propylene is a polymer where oil and solvent resistance is poor, however, it is fairly good in ketones and alcohols. It is not recommended for exposure to aromatic hydrocarbons. New Brunswick Scientific User’s Guide...

  • Page 150: Index

    Calibration of Touchscreen, 50, 97 Calibration Screen, 58 Acid Concentration, 127 Cascade Acid Type, 127 Creating a, 82 Adding a Utility Station, 98 Cascade Screen, 58, 82 Adding New Hardware, 98 CAUTION Addition Tubing Symbol for, v Size of, 79 Cautions Aeration, 6 Recap of, 2...
  • Page 151 Filling the Water Jacket, 29 International Offices Foam Control, 7, 101 List of, iii Foam Exhaust Tube Installing the, 33 Foam Level, 7 Lag Phase, 103 Foam Probe Level Probe(s) Installing the, 32 Installing the, 33 New Brunswick Scientific User’s Guide...
  • Page 152 Level Probes Control of, 6 Application of, 89 pH Probe, 6 Liquid Addition Systems, 78 Calibration of, 62 Loading Pump Tubing, 77 Inspection of, 62 Location Installation of, 34, 64 Environment, 9 Maintenance of, 65 Physical, 9 Storage of, 65 Loop Setpoints Entering the, 56 Explanation of Constants, 119...
  • Page 153 Recap of, 1 Thermowell Warranty, vi Installation of, 32 Water & Drain Connections, 15 Touchscreen Water Jacket Calibrating the, 50, 97 Filling the, 29 Troubleshooting, 114 Wetted Parts, 110 Tubing Recommendations, 126 Tubing Size, 126 New Brunswick Scientific User’s Guide...
  • Page 154 BioFlo/CelliGen 115 M1369-0050 User’s Guide...

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