AUTOCLAVABLE BIOREACTOR 2 - 7 LITER
Contents
1.2. 7 liter AUXI LIARIES October 1994 Chemostat tube The chemostat tube is used in continuous fermentation This device is designed to achieve a constant level in the reactor Liquid is taken out of the reactor through the height adjustable inner tube This inner tube is shielded from the reactor by an outer tube to avoid the influence of foam and surface irregularities on the liquid level Diameter outer tube O D 8 mm LD 6 mm Diameter inner tube O D 3 18 mm LD 1 4 mm Insertion length 156 mm 1 5 liter reactor outer tube 306 mm 7 liter reactor Z81206CH03 2 5 liter reactor M18 x 1 5 port Z81206CH07 7 liter reactor M18 x 1 5 port Sample pipe I D 1 56 mm This sample pipe is designed for the sampling of small volumes The 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 Insertion length 180 mm Z81319MB13 2 and 3 liter reactor 6 mm port Z81319MB15 2 7 liter reactor 10 mm port USER MANUAL AUXI LIARIES Autoclavable Bioreactor 2 7 liter October 1994 Drain tube The drain tube is used to take relatively large samples from the culture and to drain bio reactor after finishing the process Diameter O D 6 35 mm I D 4 53 mm Insertion length 217 mm Z81319MB14 2 5 liter reactor 10 mm port Sample pipe for screens The sample pipe for screens has a ver
3. the way you ordered it Clean all parts with 70 ethanol to remove dust or dirt from shipping When assembling the reactor make sure not to damage the threaded ports always screw inthe auxiliaries straight and by hand Do notuse tools to tighten the auxiliaries in the head plate Make sure that an O ring is present between the auxiliary and the head plate in order to ensure sterility 3 2 PREPARING FOR STERILIZATION Fill the vessel with culture medium do not exceed the total volume that is specified in chapter 1 hardware specification Be sure to leave enough volume for additions after sterilization e g inoculum separately sterilized nutrients etc Fasten the six mill nuts crosswise by hand Verify the functioning of the electrodes refill electrolyte ofthe pH electrode Insert the Level probe as far as possible into the vessel and fasten it after sterilization the position of this probe can be adjusted upward to the desired height Verify the mounting of all nipples and other auxiliaries Make connections for liquid additions air in and air out with silicone tubing or other suitable sterilizable material Use appropriate filters for air in and air out To avoid wetting of the inlet filter during sterilization use a clamp to close the tubing between the head plate and the filter Close all other connections except the air out air tight with a hose and a hose clamp Close all open tubing ends with cotton and cover the ends with s
4. Fill a sterile flask to which a sterile hose is connected aseptically with inoculum The other end of the hose should be connected to a sterile needle Turn off aeration pierce the needle through the septum and transfer the inoculum to the reactor by gravity feed or by using a pump 2 In this case a sterile syringe is filled with inoculum The needle is pierced through the septum and the inoculum is pushed into the reactor this method is suitable for inoculum volumes smaller than 100 ml Other methods of inoculation can be used if they are performed aseptically 3 7 ACTIONS DURING FERMENTATION Sampling mik To sample a fermentation broth a sample pipe is needed to which a sample system is attached Connect a syringe to the tubing that is connected to the out barb of the sample system In this tube there should be a filter or psposagie cotton AIR FILTER Pull the piston of the syringe to create a vacuum in the sample bottle As a result the fermentation broth is pulled into the sample bottle Replace the sample bottle aseptically with a sterilized empty one SYRINGE SAMPLE SYSTEM SAMPLE PIPE Additions If extra substrate is needed in the culture during fermentation it can be added as described under inoculation Make sure there is enough space left in your vessel for additions 3 8 PASTEURISATION After fermentation the broth can be pasteurized to kill the organisms To do this follow the steril
5. area for oxygen transfer High gas flow rates can be reached with open pipes or pipes with several relatively large holes All options are available for every ADI autoclavable bioreactor 2 8 USER MANUAL Autoclavable Bioreactor 2 7 liter M IXI NG AN D AE RATION October 1994 2 3 LITERATURE Bliem R and Katinger H 1988 Tibtech 6 190 195 and 224 230 Buurman C Resoort G and Plaschkes A 1986 Chem Eng Sci 41 2865 Cherry R S and Papoutsakis E T 1986 Bioproc Eng 1 29 41 Henzler H J 1982 Chem Ing Techn 54 461 Kakes E and Oosterhuis N M G 1990 A poster presentation on the Dutch Congress on Biotechnology Lavery M and Nienow A W 1987 Biotechnol Bioeng 30 368 373 Nienow A W Wisdom D J and Middleton J C 1977 2nd Eur Conf on Mixing 30 3 Cambridge England Paper F1 Oosterhuis N M G 1984 Ph D Thesis T U Delft The Netherlands Van t Riet K 1979 Ind Eng Chem Proc Des Dev 18 367 375 Voncken R M 1966 Ph D Thesis T U Delft The Netherlands Warmoeskerken M M C G 1986 Ph D Thesis T U Delft The Netherlands 2 9 USER MANUAL M IXI NG AN D AE RATION Autoclavable Bioreactor 2 7 liter October 1994 2 10 USER MANUAL Autoclavable Bioreactor 2 7 liter OPE RATION October 1994 CHAPTER 3 OPERATION 3 1 GENERAL When unpacking the equipment verify if there is any transport damage and if the reactor is complete
6. impeller blade m The impeller power number is a constant for a given system and related to the power input by the stirrer P in the following way P N prN D The mixing time in a gassed STR is as a rule of thumb twice as high as in an ungassed STR The resulting mixing time in ADI s autoclavable bioreactors for mammalian cell and bacterial culture under average operating conditions ungassed is given in the following table 2 2 USER MANUAL Autoclavable Bioreactor 2 7 liter M IXI NG AN D AE RATION October 1994 Applikon Dependable Instruments Autoclavable Bioreactors Mixing times Reactor configuration Mammalian cell culture Bacterial culture N 100rpm Marine imp N 1000rpm Rushton imp Reactor Impeller Reactor Liquid Impeller Impeller Mixing Impeller Impeller Mixing volume diameter diameter height power height time power height time v i D mm Dr mm H mm non Hmm t sec no N H mm t sec 1 45 96 150 3 28 1 3 6 11 0 2 2 45 115 125 3 28 1 6 2 45 105 175 12 11 0 2 3 45 130 200 3 28 3 2 12 11 0 4 5 60 170 200 3 37 2 8 12 15 0 3 7 60 170 300 6 37 3 4 18 15 0 4 15 74 222 365 6 45 4 3 18 15 0 5 20 74 222 550 9 45 5 7 18 19 0 8 Measurements of the mixing time in a two and three liter reactor have shown to comply well with these correlations Kakes and Oosterhuis 1990 It is obvious from this table that the mixing times are very small in relation to t
7. reactor at relatively high rotation speed gt 500 rpm see figure below Nienow et al 1977 Differences in sensitivity to damaging by the impeller and oxygen requirements between bacteria fungi and mammalian cells have led to the development of various types of impellers To improve the axially mixing characteristics of the Rushton impeller impellers with angled blades have been developed To reduce the damaging effect of the impeller the angled blades are curved in order to create a marine type of impeller for application in mammalian cell or fungi cultures This impeller also has a good axially pumping capacity which ensures mixing of the complete reactor medium and improves refreshment of the topmost layer of liquid to achieve higher oxygen transfer rates without sparging see chapter 2 2 aeration Secondary circulation loop Gas circulation patterns in aerated stirred vessels at increasing stirring speed constant gas feed 2 5 USER MANUAL M IXI NG AN D AE RATION Autoclavable Bioreactor 2 7 liter October 1994 2 2 AERATION Gas liquid mass transfer in cell culture systems is governed by the solubility of the gas in the liguid medium Bliem and Katinger 1988 its molecular diffusivity and the driving force of the gas which vary with temperature and pressure and may be described by the expression OTR kja 0 O where OTR oxygen transfer rate k liquid phase mass transfer coeff a gas liquid in
8. 7 liter reactor Z711203001 Level detector for 2 7 liter reactor 5 7 BLIND STOPPERS Blind stopper 6 10 and 12 mm ports These blind stoppers can be used to blind unused ports in the head plate the assemblies are fitted into the head plate from the inside of the reactor leaving only a flat surface in the reactor Z81321BP03 2 and 3 liter reactor 6 mm port Z81322BP03 2 7 liter reactor 10 mm port Z81322BP08 2 7 liter reactor 12 mm port Blind stopper threaded ports The following blind stoppers are available for the threaded ports Z81301BD04 2 7 liter reactor G3 4 port Z81301BD03 2 7 liter reactor M30 x 1 port Z81301BD02 2 7 liter reactor M18 x 1 5 port 5 16 USER MANUAL Autoclavable Bioreactor 2 7 liter October 1994 DRAWINGS CHAPTER 6 DRAWINGS The following drawings are enclosed D3002 1b 12 006 D3002 1 12 013 D3003 1 12 018 D3003 1 12 017 D3003 1 12 019 D3005 1a 12 001 D3005 1a 12 007 D3007 1a 12 014 D3007 1a 12 015 D3002 1a 06 005 D3007 1a 06 004 D3002 1 06 006 D3007 1 06 003 Bioreactor 21 dished H D 1 5 Bioreactor 21 jacketed H D 1 5 Bioreactor 31 flat bottom Bioreactor 31 dished bottom Bioreactor 31 jacketed Bioreactor 51 dished bottom Bioreactor 51 jacketed Bioreactor 71 dished bottom Bioreactor 71 jacketed Stirrer assembly lipseal 2 5 liter Stirrer assembly lipseal 7 liter Stirrer ass magn coupled 2 5 liter Stirrer ass magn coupled 7 liter 6 1
9. E FOR LEVEL CONTROL 06 3x 010 6x 612 2x M18x1 5 5x The following ports are present one central M30 x 1 port for the stirrer assembly one G3 4 port for dissolved oxygen sensor five M18 x 1 5 ports for auxiliaries like other sensors pH mV etc two 12 mm ports for auxiliaries like harvesting pipes etc six 10 mm ports for auxiliaries like air inlet pipes etc three 6 mm ports for baffles etc 1 7 USER MANUAL HARDWARE SPECIFICATION Autoclavable Bioreactor 2 7 liter October 1994 The 5 and 7 liter reactor 02 CONTACT HOLE FOR LEVEL CONTROL M18x1 5 5x 10 10x 012 2x MILL NUT 6x The following ports are present one central M 30 x 1 port for the stirrer assembly one G3 4 port for the dissolved oxygen sensor five M 18 x 1 5 ports for auxiliaries like other sensors pH mV etc two 12 mm ports for auxiliaries like harvesting pipes etc ten 10 mm ports for auxiliaries like baffles air inlet pipes etc Caution It is strongly advised to ground the head plate in order to avoid disturbances on electrode sensor signals static electricity use one of the two 2 mm holes in the head plate The other 2 mm hole can be used for level control connection USER MANUAL Autoclavable Bioreactor 2 7 liter HARDWARE SPECIFICATION October 1994 1 4 THE STIRRER ASSEMBLY Two different stirrer assembly types are available a
10. additions to or sampling from the culture fluid can take place at any level inside the reactor Z811302015 2 7 liter reactor M18 x 1 5 port Assembly holder 6 mm tube for 10 mm port This device can be inserted into a 10 mm port and can be used to hold any 6 mm O D tube The insertion length of this tube can be varied additions to or sampling from the culture fluid can take place at any level inside the reactor Z81320AHO00 2 7 liter reactor 10 mm port 5 1 AUXILIARIES Sample pipe fixed length This assembly is used to sample the culture fluid Tube diameter O D 6 mm to fit in a 10 mm port 9 5 mm to fit in a 12 mm port Insertion length 213 mm for 2 3 and 5 l reactors 325 mm for 7 1 reactor Z81319MB03 2 5 liter reactor 10 mm port Z81319MB05 2 5 liter reactor 12 mm port Z81319MB07 7 liter reactor 10 mm port Z81319MB08 T liter reactor 12 mm port USER MANUAL Autoclavable Bioreactor 2 7 liter October 1994 Sample pipe height adjustable The height adjustable sample pipe assembly consists of an assembly holder for 6 mm tubes and a O D 6 mm sample tube Tube diameter 1 D 4mm Maximum insertion length 232 mm 2 5 liter reactor 320 mm 7 liter reactor With this device the culture fluid can be sampled at any desired level Z81319MB04 2 5 liter reactor 10 mm port Z81319MB06 7 liter reactor 10 mm port 5 2 USER MANUAL Autoclavable Bioreactor 2
11. at can be reached in practice is upto 10 times higher Kakes and Oosterhuis 1990 Sparging however is known to cause inadmissible shear stress for mammalian cells will increase the problem of foam formation in bacterial as well as mammalian cultures and is therefore limited in its applications Applikon Dependable Instruments Autoclavable Bioreactors Theoretical oxygen transfer rates Reactor configuration Mammalian cell culture Bacterial culture N 1000 N 100 rpm Marine rpm Rushton impellers Aeration impellers Aeration 1 Reactor Impeller Reactor Impeller Oxygen transfer Impeller Oxygen transfer volume diameter diameter coalesc non coal D mm Dr mm 1 45 96 3 1 18 6 7 33 2 45 115 3 1 12 2 45 105 12 8 37 3 45 130 3 1 9 12 7 27 5 60 170 3 2 18 12 10 52 7 60 170 6 2 24 18 12 59 15 74 222 6 3 31 18 16 75 20 74 222 9 4 36 18 16 65 Since the required k a value and admitted aeration flow vary widely from system to system various ways are used to introduce a gas flow into the system For mammalian cell cultures mostly an oxygen enriched air overlay is used in combination with an axially pumping impeller In order to get a large surface area and thus improve the oxygen transfer reactors with a low H D value usually 1 are used for this application Fermentations that require both a low stirrer speed and gas flow need a sintered steel sparger in order to create small bubbles and thus a large
12. ature measurement and control is very important in bio technology The thermometer pocket allows you to insert a temperature probe Pt100 or mercury thermometer in the reactor Fill the thermometer pocket with water or silicone oil in order to improve thermal contact between the culture and the probe Insertion length 200 mm 2 5 liter reactor 300 mm 7 liter reactor Z81323TP03 2 5 liter reactor 10 mm port Z81323TPO7 7 liter reactor 10 mm port DO electrode holder Every Applikon 2 7 liter head plate is equipped with a G3 4 port for the DO electrode holder The electrode is height adjustable Z811303001 2 7 liter reactor G3 4 port 5 15 USER MANUAL AUXI LIARIES Autoclavable Bioreactor 2 7 liter October 1994 Nipple ID 12 mm for G3 4 port This nipple can be used to fit a pH or mV electrode or any other device with an OD of 12 mm height adjustable Z81300N005 1 7 liter reactor G3 4 port Available sensors Z71201AP10 Sensor pH L 235 mm for 2 5 liter reactor Z71201AP20 Sensor pH L 385 mm for 7 liter reactor Z71201AG10 Sensor pH gel L 235 mm for 2 5 liter reactor Z71201AG20 Sensor pH gel L 385 mm for 7 liter reactor Z71202AP10 Sensor DO L 215 mm for 2 5 liter reactor Z71202AP20 Sensor DO L 215 mm for 7 liter reactor Z71203MV02 Sensor redox L 320 mm Z71204T002 Sensor temperature L 200 mm Z71205AF03 Sensor foam for 2
13. ber 1994 Baffle assembly Baffles are used to increase the mixing efficiency without baffles the medium flow can become laminar causing poor mixing efficiency and mass transfer The baffles are mounted near the reactor wall for optimum mixing performance The baffle assembly consists of one baffle and mounting material Normally three baffles are used to create maximum mixing efficiency Insertion length 220 mm for 2 and 3 liter reactor 223 mm for 5 liter reactor 317 mm for 7 liter reactor Z81326KS03 2 and 3 liter reactor 6 mm port Z81326KS05 5 liter reactor 10 mm port Z81326KS07 7 liter reactor 10 mm port Draught tube The draught tube is used to obtain maximum mixing efficiency and optimum oxygen mass transfer by enforcing an axial flow pattern in the reactor The draught tube must be used in combination with a marine impeller the vortex type for foam killing the scoping type in case a vortex or gas bubbles in the culture liquid are not allowed Insertion length 220 mm 3 liter reactor Tube length 120 mm Draught tubes for other reactors on request Z81334DT03 3 liter reactor 3 x 6 mm port 5 12 USER MANUAL Autoclavable Bioreactor 2 7 liter AUXI LIARIES October 1994 Impellers The following impellers are available for the Applikon stirrer assemblies lipseal and magnetically coupled Z81312RS02 Rushton impeller 4 bladed 2 3 liter reactor Ma Z81313R602 Rushton impeller 6 blade
14. clavable BI ctober 1994 AUTOCLAVABLE BIOREACTOR 2 7 LITER USER MANUAL V3UBCE0061 USER MANUAL Autoclavable Bioreactor 2 7 liter CONTE NTS October 1994 TABLE OF CONTENTS Chapter Description Page 1 Hardware specification 1 1 1 1 General 1 1 1 2 The reactors 1 2 1 3 The head plates 1 7 1 4 The stirrer assemblies 1 9 2 Mixing and aeration 2 1 2 1 Mixing 2 1 2 2 Aeration 2 6 233 Literature 2 9 3 Operation 3 1 3 1 General 3 1 3 2 Preparing for sterilization 3 1 3 3 Sterilization 3 2 3 4 Installation 3 2 3 5 Preparing for operation 3 2 3 6 Inoculation 3 3 3 7 Actions during fermentation 3 3 3 8 Pasteurization 3 3 4 Maintenance 4 1 4 1 The reactor 4 1 4 2 The stirrer assembly 4 2 5 Auxiliaries 5 1 5 1 Sampling 5 1 5 2 Aeration 5 6 5 3 Addition 5 9 5 4 Mixing 5 11 5 5 Heat exchangers 5 14 5 6 Sensor holders 5 15 5 7 Blind stoppers 5 16 6 Drawings 6 1 USER MANUAL Autoclavable Bioreactor 2 7 liter HARDWARE SPECIFICATION October 1994 CHAPTER 1 HARDWARE SPECIFICATION 1 1 GENERAL The stirred tank reactors STR are the most applied reactor types in biotechnology The Applikon 2 7 liter autoclavable bioreactors offer several advantages its modular and flexible design requires minimum bench space and is very practical in operation In combination with Applikons stirrer assemblies motors impellers and auxiliaries you can configure the reactor for a variety of applications like medium optim
15. d 2 3 liter reactor Z81313R645 Rushton impeller 6 bladed vortex 2 3 liter reactor Z81313R607 Rushton impeller 6 bladed 5 and 7 liter reactor Z81314RCO02 Marine impeller vortex 2 3 liter reactor Z81314RC03 Marine impeller scoping 2 3 liter reactor Z81314RCO07 Marine impeller vortex 5 and 7 liter reactor Z81314RC08 Marine impeller scoping 5 and 7 liter reactor 5 13 USER MANUAL AUXI LIARIES Autoclavable Bioreactor 2 7 liter October 1994 5 5 HEAT EXCHANGERS Two different types of heat exchangers are available 1 fitting into a M18 x 1 5 port This heat exchanger can be used in the 2 5 liter reactors Insertion length 226 mm Z81317KV03 2 5 liter reactor M18 x 1 5 port 2 fitting two 10 mm ports This type of heat exchanger is available for the 5 and 7 liter reactor Insertion length 210 mm 5 liter reactor 315 mm 7 liter reactor Z81317HE05 5 liter reactor two 10 mm ports Z81317HE07 7 liter reactor two 10 mm ports 5 14 USER MANUAL Autoclavable Bioreactor 2 7 liter AUXI LIARIES October 1994 5 6 SENSOR HOLDERS pH mV nipple This nipple fits into a M18 x 1 5 port and can accommodate a pH or mV electrode a liquid entry system the glass condenser or other 12 mm O D tubes All items that are hold by this nipple are height adjustable Z81300N002 2 7 liter reactor M18 x 1 5 port Thermometer pocket Temper
16. er October 1994 The 31 jacketed bioreactor Z61103CT04 This reactor has the same specifications as the 3 1 dished bottom reactor however temperature control is performed through the glass jacket In this way the temperature can be controlled more accurately the jacket can both be used for heating and cooling Internal jacket volume 1 31 The 5 1 dished bottom bioreactor Z61 1000005 Specifications Max working volume 4 81 H D ratio 1 6 Min working volume 21 Working volume 3 41 H D ratio 1 1 For temperature control a heat exchanger inside the reactor can be used or a silicone heating blanket can be wrapped around the vessel The reactor comes with a tripod for support USER MANUAL Autoclavable Bioreactor 2 7 liter October 1994 The 5 jacketed bioreactor Z61104CT05 This reactor has the same specifications as HARDWARE SPECIFICATION the 5 1 dished bottom reactor however temperature control is performed through the glass jacket Jacket volume 2 81 The jacketed reactor is especially developed for mammalian cell cultures the large heat transfer area ensures optimum conditions for temperature control This reactor has a tripod for support The 7 dished bottom bioreactor Z611000007 Specifications Max working volume 6 91 H D ratio 2 2 Min working volume 2 01 Working volume 5 41 H D ratio 1 8 F
17. er X X X clockwise Pull off the lower part be aware of the magnetic field Remove the motor coupling part from F the head plate by turning it counter clockwise Dismantle the motor coupling part by unscrewing the 4 slotted cheese head screws and unscrew the pillars C Remove the spring pin D from the shaft and remove the upper lipseal 7077770000070 E Remove the top plate F Pull the stirrer shaft and the inner magnets out of the housing Clean all parts with tissue paper G lubricate the ball bearings G with the special grease art no V3LA120031 A Remove the stirrer shaft from the rotor section clean and lubricate the surface of the ball bearing G Replace any O ring or lipseal if necessary and reassemble the stirrer assembly carefully N OTZA ZZZZZZA y i ZZ Q e It LLLLL 4 3 USER MANUAL MAINTENANCE Autoclavable Bioreactor 2 7 liter October 1994 4 4 USER MANUAL Autoclavable Bioreactor 2 7 liter AUXI LIARIES October 1994 CHAPTER 5 AUXILIARIES In this chapter the head plate auxiliaries for the 2 7 liter bio reactors are listed 5 1 SAMPLING Assembly holder 6 mm tube for M18 x 1 5 port This device fits into a M18 x 1 5 port and can be used to hold any 6 mm O D tube The insertion length of this tube can be varied
18. he characteristic times that are to be expected for the metabolism e g oxygen uptake rate substrate consumption rate of the micro organisms Therefore the medium can be looked upon as being ideally mixed at all times during fermentation USER MANUAL M IXI NG AN D AE RATION Autoclavable Bioreactor 2 7 liter October 1994 As the bubbles are coalescing behind the impeller and broken up by vortices created by the impeller the gas will be homogenised in a similar way as the medium and its components The influence of gas feed on this mechanism is illustrated in the figure below Warmoeskerken 1986 Despite this mixing of the gas phase the cavities behind the impeller blades will decrease the power input to the reactor and thus decrease the oxygen transfer rate to the reactor medium see chapter 2 2 aeration disk direction of blade direction of rotation rotation vortex cavity clinging cavity direction of blade rotation large cavity Cavity shape behind Rushton type impellers for small medium and high gas feed rates USER MANUAL Autoclavable Bioreactor 2 7 liter M IXI NG AN D AE RATION October 1994 The operating range of the impeller speed is limited due to the potential damaging effect of the rotating impeller on the micro organisms Therefore it may be impossible to satisfy the criteria for homogeneity Furthermore a Rushton type of impeller will only create a homogeneous distribution of bubbles throughout the
19. ily operated sampling device Z81207SS02 2 7 liter reactor 5 5 USER MANUAL AUXI LIARIES Autoclavable Bioreactor 2 7 liter October 1994 5 2 AERATION Air inlet pipe 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 reguired 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 Insertion length 236 mm 2 5 liter reactor 330 mm 7 liter reactor Z81318L002 2 liter reactor 10 mm port Z81318L003 3 liter reactor 10 mm port Z81318L005 5 liter reactor 10 mm port Z81318L007 7 liter reactor 10 mm port Air inlet pipe with porous sparger In cell culture fermentations high gas flow rates causing shear forces might damage the cells To be able to meet the oxygen demand of the cells at lower gas flow the exchange surface must be increased This can be achieved by using this sparge pipe The sintered metal tip produces tiny air bubbles for optimum gas distribution Insertion length 238 mm 2 5 liter reactor 330 mm 7 liter reactor Z81318L004 2 liter reactor 10 mm port Z811303005 3 liter reactor 10 mm port Z81318L006 5 liter reactor 10 mm port Z81318L008 7 liter reactor 10 mm port Z8 11303008 Porous sparger for air inlet pipe 5 6 USER MANUAL Autoclavable Bioreactor 2 7 li
20. ization screening of strains product optimization scale up down studies reactor optimization continuous cultures perfusion systems etc Danger Glass is a material that may be damaged easily scratches on the surface As a result its strength is reduced Therefore do not apply a process pressure that exceeds 50 kPa 0 5 bar 7 psig Reduce the pressure of the inlet gasses to this extend or apply a pressure relief that is properly adjusted 1 1 USER MANUAL HARDWARE SPECIFICATION Autoclavable Bioreactor 2 7 liter October 1994 1 2 THE REACTORS The 2 7 liter bioreactors are ideals tool for researchers who start fermentation studies and for those applications where small or medium volumes are required Due to the modular design the reactors can be used for a variety of applications including microbial and yeast fermentations cell cultures etc Reactor material borosilicate glass Other materials in contact with the medium stainless steel head plate silicone rubber viton and EPDM The reactor types and their specifications are listed below The21ldished bottom bioreactor 2611020002 Specifications Maximum working volume 2 21 H D ratio 2 3 Minimum working volume 0 51 Working volume 1 71 H D ratio 1 9 For temperature control a heat exchanger inside the reactor can be used or a silicone heating blanket can be wrapped around the vessel The reactor comes with a
21. ization procedure however the operating temperature of the autoclave should now be about 80 C After pasteurization the head plate of the reactor can be removed and the fermentation broth can be collected 3 3 USER MANUAL OPERATION Autoclavable Bioreactor 2 7 liter October 1994 3 4 USER MANUAL Autoclavable Bioreactor 2 7 liter MAI NTE NANCE October 1994 CHAPTER 4 MAINTENANCE 4 1 THE REACTOR AND HEAD PLATE After finishing the fermentation process the glass and stainless steel parts should be cleaned thoroughly Use hot water 70 ethanol or other suitable cleaners to clean all parts Never use abrasive materials to clean the metal parts After cleaning dry the parts and reassemble the reactor Take care not to damage or forget any O rings since this can cause contamination during the next run When the reactor is used frequently it is advised to replace the O rings of the auxiliaries twice a year Note Make sure that the pH electrode is cleaned thoroughly before it is stored refer to the user manual of this electrode 4 1 USER MANUAL MAINTENANCE Autoclavable Bioreactor 2 7 liter October 1994 4 2 THE STIRRER ASSEMBLY The stirrer assembly should be disassembled cleaned and lubricated twice a year Disassembling instructions are listed below 4 2 1 LIPSEAL STIRRER ASSEMBLY Remove the head plate from the AP En reactor and remove the impellers from Y 7 Be the stirre
22. jacket with water to improve the heat transfer connect and close the lower tube fitting do not close the upper one 3 4 INSTALLATION Put the reactor as near as possible to the control equipment the Bio Processor or Bio Controller and connect all electrodes to this equipment Connect the heat exchanger to the thermo circulator hook up the air inlet pipe to the flow console and if present connect cooling water to the condenser Connect all sterile fluids acid base etc that have to be added to the medium aseptically to the inlet pipes To improve heat transfer between the thermometer pocket and the Pt100 sensor fill the thermometer pocket with water or silicone oil This will decrease the dead time of the sensor and will make your temperature control more accurate When operating at higher temperature silicone oil has the advantage of a lower vapour pressure less evaporation 3 5 PREPARING FOR OPERATION After connecting all cables and tubing adjust the setpoints of the controllers to the desired value temperature pH DO etc Switch on the thermo circulator stirrer motor acid and or base pumps and gas flow When temperature pH etc have reached their setpoint and are stabilized the bioreactor is ready for inoculation USER MANUAL Autoclavable Bioreactor 2 7 liter O P E RATIO N October 1994 3 6 INOCULATION There are several ways to inoculate Two methods that are commonly used are described here 1
23. lipseal stirrer assembly and a magnetically coupled stirrer assembly Z81315R003 Lipseal stirrer assembly 2 3 5 liter Z81315R007 Lipseal stirrer assembly 7 liter Z81315MG03 Magnetically coupled stirrer assembly 2 3 5 liter Z81315MG07 Magnetically coupled stirrer assembly 7 liter Material stainless steel 316 The assemblies are autoclavable and are designed for long periods of operation with minimum maintenance a X KI X XK XxX X Lipseal stirrer assembly Magn coupled stirrer assembly ex shaft The stirrer shaft of the lipseal stirrer assembly is coupled directly to the stirrer motor This is the most common way of coupling whereby contamination free operation is ensured by the use of viton lipseals The magnetically coupled stirrer assembly is especially developed for applications concerning genetic engineering organisms and cell culture This ensures absolute contamination free operation since there is no moving seal between the reactor content and the environment The applicable stirrer motors are described in the stirrer motor manual 1 9 USER MANUAL HARDWARE SPECIFICATION Autoclavable Bioreactor 2 7 liter October 1994 1 10 USER MANUAL Autoclavable Bioreactor 2 7 liter October 1994 2 1 CHAPTER 2 MIXING AND AERATION For an optimum performance ofany biological system itis necessary to kee
24. n the range of 5 50 ml O I h at 37 C and atmospheric pressure Therefore the required k a for a typical batch culture operation is likely to be 0 5 5 10 s Static liquid surface aeration is insufficient even for these low reguirements see table on previous page For high density cultivation as reached in long term perfusion systems the required ka may increase to as much as 0 1 1 10 s this oxygen requirement can only be supplied by sparging Sparging is the most efficient way to obtain high k a values Most experimental data for kja values can be estimated by the correlations of Van t Riet 1979 and Henzler 1982 adapted for temperature difference for coalescing media ka 1 022 0 026 P V v for non coalescing media ka 1 022 0 016 P V v where T temperature C P the gassed power input by impeller N m s volume m V superficial gas velocity m 57 At very low aeration rates P will be equal to the ungassed power input P Lavery and Nienow 1987 but at higher aeration rates the gassed power input has to be calculated according to 3 P 0 5 P 0 5 N pr ND 2 7 USER MANUAL Autoclavable Bioreactor 2 7 liter October 1994 MIXING AND AERATION The theoretical k a values from these correlation for ADI s autoclavable reactors are given in the table below Measurements of kja values on a two and three liter scale have shown that the oxygen transfer capacity th
25. or temperature control a heat exchanger inside the reactor can be used or a silicone heating blanket can be wrapped around the vessel The reactor comes with a tripod for support 1 5 HARDWARE SPECIFICATION The 7 1 jacketed bioreactor Z61103CT07 This reactor has the same specifications as the 7 1 dished bottom reactor however temperature control is performed through the glass jacket Jacket volume 4 21 The jacketed reactor is especially developed for mammalian cell cultures the large heat transfer area ensures optimum conditions for temperature control This reactor has a tripod for support tripod of the 5 liter reactor with extension shafts sA USER MANUAL Autoclavable Bioreactor 2 7 liter October 1994 USER MANUAL Autoclavable Bioreactor 2 7 liter HARDWARE SPECIFICATION October 1994 1 3 THE HEAD PLATES The head plates are made of stainless steel 316 The port lay outs are presented in the figures below The 2 liter reactor MILL NUT 6x M18x1 5 5x CONTACT HOLE FOR LEVEL CONTROL G 3 4 06 3x 910 6x The following ports are present one central M 30 x 1 port for the top stirrer one G3 4 port for dissolved oxygen sensor five M 18 x 1 5 ports for auxiliaries three 6 mm ports for baffles or other auxiliaries six 10 mm ports for auxiliaries The 3 liter reactor MILL NUT 6x 02 CONTACT HOL
26. p the environment of the micro organisms at optimal conditions Apart from temperature and medium composition the two most important factors that effect this environment are the degree of mixing and aeration MIXING The aim of mixing is to obtain uniform conditions in the working volume of the bioreactor in order to obtain an optimal mass transfer to avoid gradients of any of the medium components and to keep the microcarriers or cells in suspension In a normally used stirred tank reactor mixing is accomplished by the impeller The resulting flow pattern is a function of the impeller configuration agitation speed the geometry of the system and gas inflow rate employed One aspect of mixing is preventing the cells or microcarriers to settle In order to achieve this the fluid velocity must at least be equal to the settling velocity of the particles Vu which can be calculated according to Stokes law Cherry and Papoutsakis 1986 Vett d p pr g 18 7 where d particle diameter m Ps particle density kg m Pr medium density kg m g gravitational constant m s n medium viscosity N m s Stokes law only holds for particles whose Reynolds numbers are smaller than 1 which holds for all normally used biological systems As the biomass should be homogeneously distributed throughout the reactor not only must the particles be lifted from the bottom of the vessel but they must also be transported th
27. r shaft Screw the stirrer Y 7 A assembly out of the head plate Y Y Remove the coupling ring A at the NIN top of the assembly by unscrewing the NR four slotted cheese head screws Screw out the pillars B on which the coupling ring was mounted and remove the spring pin C and the upper lipseal D Take off the upper cover E of the stirrer assembly Remove the lower cover H by loosening the three small screws and C D Eo removing the lipseal A E EN EY E F G TO Remove the circlip G from the shaft on top of the ball bearing F Turn the stirrer assembly upside down and gently push the shaft out of the ball bearing house This is most easily achieved by placing the top of the stirrer shaft on the table upside down and pressing the housing down z ga mi 227 Cleanall parts and lubricate the ball LE ANPR eee sees bearing again with special grease art AE HE 7 A ENZA N no V3LA120031 Replace any O ring or lipseal if necessary and reassemble the stirrer assembly carefully 4 2 USER MANUAL Autoclavable Bioreactor 2 7 liter MAI NTE NANCE October 1994 4 2 2 MAGNETICALLY COUPLED STIRRER ASSEMBLY Remove the head plate from the reactor and put it upside down on the motor coupling ring of the stirrer assembly i Turn the dismantling screw A as far as possible into the housing Loosen the lower part of the stirrer assembly i by turning the rotor B count
28. rough the whole volume of the reactor 2 1 MIXING AND AERATION USER MANUAL M IXI NG AN D AE RATION Autoclavable Bioreactor 2 7 liter October 1994 Generally the minimum required stirring speed to achieve homogeneous suspension N s is much greater than that to lift the particles from the bottom of the reactor Buurman et al 1986 also derived a simple Froude relationship to define homogeneity in a stirred tank reactor STR based on the assumption of fluctuating velocity being proportional to the circulation velocity PrN D 8 4 p d constant where D diameter of the impeller m which implies that Ni L D The parameter used to describe the medium homogeneity aspect of mixing is the mixing time which is in fact also the characteristic time for mixing The mixing time t is often expressed in the liquid circulation time t In the case of a stirred tank t equals 4 times t Voncken 1966 The liquid circulation time in STRs with a Rushton impeller can be calculated according to Oosterhuis 1984 t V 0 75 ND where V working volume of the reactor m N rotation speed of the impeller s If the geometry of the system has to be taken into account in ungassed STRs the following correlation for the mixing time can be used ta 1 2 N D D H D N D Hy where D diameter of the reactor m H liguid height in the reactor m N impeller power number H height of
29. rs glass condenser fits into the pH mV nipple stainl steel condenser fits into the M18 x 1 5 port Z81309L003 glass condenser for 2 5 liter reactor pH mV nipple Z81309L002 SS condenser for 2 5 liter reactor M18 x 1 5 port Z81309L007 SS condenser for 7 liter reactor M18 x 1 5 port 5 8 USER MANUAL Autoclavable Bioreactor 2 7 liter AUXI LIARIES October 1994 5 3 ADDITION Septum holder The septum holder is eguipped with a silicone rubber septum and can be used as a universal addition port by piercing it with one or more needles Z81302PD02 2 7 liter reactor M18 x 1 5 port Needle for septum The needle is used to pierce the septum and to add a fluid or gas to the culture Z81309IN02 Addition pipe 10 mm port This addition pipe can be used to add fluids or gasses to the reactor Z81324MT02 2 7 liter reactor 10 mm port 5 9 USER MANUAL AUXI LIARIES Autoclavable Bioreactor 2 7 liter October 1994 Medium inlet triple The medium inlet triple allows you to eguip one M18 x 1 5 port with three addition ports 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 2 7 liter reactor M18 x 1 5 port Liquid entry system When running a continuous culture backgrowth of organisms into the medium container mus
30. t 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 mV nipple Z81300N002 Z81309IN03 2 7 liter reactor pH mV nipple Liquid addition bottle The liquid addition bottles are available in the following sizes 0 5 liter Z811302009 1 0 liter Z811302010 2 0 liter Z811302011 5 0 liter Z811302012 10 liter Z8 11302013 20 liter Z811302014 The liquid addition bottle comes with an air inlet filter 5 10 USER MANUAL Autoclavable Bioreactor 2 7 liter AUXI LIARIES October 1994 5 4 MIXING Two types of stirrer assemblies are available for insertion in the central M30 x 1 port of the head plate 1 The lipseal stirrer assembly Material Stainless Steel Diameter shaft 8 mm Z81315R003 2 5 liter reactor M30 x 1 port Z81315R007 7 liter reactor M30 x 1 port 2 The magnetically coupled stirrer assembly zi n Material Stainless Steel Diameter shaft 8 mm Xx KR r Z81315MG03 2 5 liter reactor M30 x 1 port Z81315MG07 7 liter reactor M30 x 1 port 5 11 USER MANUAL AUXI LIARIES Autoclavable Bioreactor 2 7 liter Octo
31. ter AUXI LIARIES October 1994 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 a 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 listed below Z811302020 1 7 liter reactor Pressure relief valve When over pressure is applied in the glass Applikon 1 7 liter bio reactors 1t is advised to install this relief valve The pressure at which the relief valve will open can be adjusted manually Z811302050 1 7 liter reactor M18 x 1 5 port Air outlet pipe The air outlet pipe can be used for either gas outlet or gas overlay The latter use is for head space aeration separate from or in combination with sparging gas through the culture Z81308LU02 2 7 liter reactor 10 mm port 5 7 USER MANUAL AUXI LIARIES Autoclavable Bioreactor 2 7 liter October 1994 Air outlet condenser Working at elevated temperature and using aeration of the culture might cause too much evaporation during fermentation increase of nutrient concentration and decrease in volume this can be prevented by using an air outlet condenser Available condense
32. terfacial area per unit liquid volume o liquid phase oxygen concentration in eguilibrium with the bulk gas phase at 1 atm and 37 C 0 18 mmol O A O actual oxygen concentration of the liquid phase The term ka represents the volumetric overall mass transfer coefficient and is one of the most common parameters used to describe the efficiency of an aeration system Bliem and Katinger 1988 compared the typical efficiency of some forms of aeration which are given in the following table Efficiencies of different aeration systems Typical oxygen transfer rates k a 57 Aeration system under culture conditions using air cm s 10 10 static liquid surface aeration 10 10 10 10 stirred liquid surface aeration 10 8 10 10 10 silastic membrane aeration 5 10 3 10 10 3 10 dynamic wire mesh with agitation 7 10 sparged and stirred liguid for cell culture 10 7 10 sparged and stirred liquid for microbial culture 2 6 USER MANUAL Autoclavable Bioreactor 2 7 liter M IXI NG AN D AE RATION October 1994 The kja values are strongly influenced by the medium composition For example the addition of silicone antifoam can cause the ka to decrease drastically whereas the addition of salts to the medium will increase the ka Lavery and Nienow 1987 Bliem and Katinger 1988 For a mammalian cell culture to a density of 5 10 cells l a typical batch culture the oxygen requirement is i
33. terilizable foil or paper Make sure that the vessel is not completely closed since pressure differences during sterilization may damage the reactor or the probes Use the air outlet filter to maintain pressure equilibrium in and outside the reactor The heat exchanger should be empty during sterilization 3 1 USER MANUAL OPERATION Autoclavable Bioreactor 2 7 liter October 1994 3 3 STERILIZATION The bioreactor with all accessories except the stirrer motor can be placed in an autoclave The space needed in the autoclave can be derived from the drawings that are enclosed in the English copy of this manual The autoclave should stay at 121 C for at least 20 minutes in order to kill all organisms and thermo resistant spores After sterilization let the autoclave cool down without opening it until the temperature is below 90 C When the temperature in the autoclave has dropped below 90 C it can be opened to allow it to cool down further This cooling procedure should be performed to avoid low pressure in any part of the reactor system Low pressure in tubing might result in contamination when the tube is connected to a peripheral device Low pressure in a pH electrode may result in sudden boiling of the electrolyte Note In case of a jacketed reactor the sterilization interval might need to be increased since the empty jacket has a poor heat transfer capacity If the medium cannot stand a longer interval you can fill the
34. tripod for support The 21 jacketed bioreactor Z61104CT04 Specifications Maximum working volume 2 21 H D ratio 2 3 Minimum working volume 0 51 Working volume 1 71 H D ratio 1 9 Jacket volume 1 31 The jacketed reactor is especially developed for mammalian cell cultures the large heat transfer area ensures optimum conditions for temperature control The tripod is supplied with extension shafts USER MANUAL Autoclavable Bioreactor 2 7 liter HARDWARE SPECIFICATION October 1994 The 3 1 flat bottom bioreactor Z611000004 This reactor is generally used for bacterial fermentations with micro organisms that are not very sensitive to shear forces Specifications Max working volume 3 21 H D ratio 1 8 Min working volume 0 441 Working volume 2 61 H D ratio 1 5 A heat exchanger inside the vessel or a silicone heating blanket wrapped around the vessel can be used for temperature control The 3 dished bottom bioreactor Z61101C006 This reactor is mostly used for culturing shear sensitive cells organisms The dished bottom ensures optimum mixing properties Specifications Total volume 3 21 H D ratio 1 9 Minimum working volume 0 47 ml Working volume 2 71 H D ratio 1 5 Temperature control can be obtained in the same way as with the 3 1 flat bottom bioreactor 1 3 USER MANUAL HARDWARE SPECIFICATION Autoclavable Bioreactor 2 7 lit
35. y 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 see below TTT TTT TTT 1 E Z81319MB09 2 5 liter reactor 10 mm port Z81319MB11 7 liter reactor 10 mm port Sample screen Available pore sizes Z811303010 13 u sample screen Z811303011 25 u sample screen Z811303012 76 u sample screen Z811303013 105 u sample screen 5 4 USER MANUAL Autoclavable Bioreactor 2 7 liter AUXI LIARIES October 1994 Sample pipe I D 10 mm This sample pipe is designed for sampling cultures with flocculating organisms in this case a sample pipe with a small diameter will ruin the flocks and the pipe will be clogged The shear forces inside this sample pipe are nearly negligible The sample pipe can be fitted in a 12 mm LD pH mV nipple Z81319MB22 2 5 liter reactor pH mV nipple Z81319MB23 7 liter reactor pH mV nipple Sample system The sample system with a 60 or 30 ml glass bottle can be mounted onto the head plate of the bio reactor This system completed with a syringe and connected to the sample pipe tubing provides your bio reactor with an eas
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