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STANDARD OPERATING PROCEDURE

Fermentor, 5-LModel: New Brunswick, Celligen Model 310

Manufacturer: Eppendorf North America

Location: Fermentation Facility, 1621 Food Sciences Building

Publication Date: 03/06/2014

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Description and Uses

The CelliGen 310 runs on a BioFlo 310 cabinet. It can be used for batch, fed-batch or continuous culture with microprocessor control of pH, dissolved oxygen (DO), agitation, temperature, pump feed, antifoam, foam/level and additional analog/digital inputs and outputs.

Power Specifications

Motor: CelliGen Model 310

Voltage/Amperage: 100-120V/15A

Speed/Frequency: Variable speed control@50-60 Hz.

For further information on power specifications, please see page 15 in Operations Manual (#M1284-0056) located in 1621 Food Sciences Building.

Potential Hazards and Safety Precautions

Electric Shock/Standard Voltage (100-120V)

• Make sure that the wall outlet receptacle is properly wired and grounded, and matches the instrument’s power cord and plug.

• Do not touch the power cord or plug if hands or feet are wet, or if standing on a wet/damp surface, as severe electrical shock or death may result.

• Never attempt to clean, move or adjust mixer while it is running or while it is connected to a power source.

Excessive Weight/Possible Damage to Hands/Fingers when Moving or Repositioning

• The fermentor is heavy. If available, wear shoes with steel caps when carrying the fermentor to prevent injury if the fermentor is dropped. Also, use extreme caution when handling or moving the fermentor, as the vessel is made of glass.

Combustibility and Explosion/Possible Physical Damage or Injury

• Do not use the mixer with materials capable of developing flammable or explosive vapors.

Biohazard/Possible Infection from use of Pathogenic Microbes

• Normal operation involves the use of many types of microorganisms, all of which can be opportunistic pathogens. Occasional use may also involve working with genetically modified microbes. Use extreme caution and good sterile techniques when working with these organisms. After use, autoclave the fermentor to kill any remaining microbes.

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Required Personal Protective Equipment

Lab Coat

Hair Net

Safety Glasses or Goggles

No Open-toed or Open-heeled Shoes

Insulated Rubber Gloves

Steel-toed Shoes are recommended

Required Training

*Denotes courses offered online

Machine & Site-Specific Training

Fire Safety & Extinguisher Training*

Laboratory Safety: Core Concepts*

Laboratory Safety: Spill Procedures

Bloodborne Pathogens and Sharps Safety*

Operation

Operation: Start-up

1. The Instruction Manual provides a more complete description of the CelliGen Model 310 Fermentor and the BioFlo 310 Control Module. Be sure to read the manual before operating the fermentor to become familiar with its correct operation and individual component parts.

2. Inspect the fermentor to see if it is clean. If not, remove the top plate by loosening the four retaining screws. Clean the vessel with warm soapy water. Then, rinse with tap water and distilled water. Use extreme caution when cleaning the vessel, as it is made of glass. Invert the clean vessel on paper towels and allow it to dry.

3. If necessary, clean the underside of the cover plate, the impeller shaft and paddles.

4. Remove the sparge tube and siphon tube from the cover plate. As they may contain remnants from the last fermentation (e.g. dry media, etc.), check to see if they are clean. If not, clean the tubes with warm soapy water and pipe cleaners. Then, rinse with distilled water. Also, run distilled water thru the sparge tube and check to see if the tube orifices are clear. If not, clean them with a piece of thin wire. Allow the tubes to dry.

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5. Remove the sampling tube from the cover plate. Check the condition of the tube and the sampling valve. These can also contain remnants of the last fermentation that must be washed out. Clean the tube and valve as described above. If the valve is especially dirty, it may be necessary to disassemble it to clean it properly. Reassemble the valve. Then, rinse the tube and valve with distilled water. Allow sufficient time for the tube and valve to dry.

6. Check the condition of the septum on the cover plate of the fermentation vessel. If necessary, replace it. The septum is a self-sealing rubber closure through which inocula, sterile media, etc. can be added to the fermentor using sterile needles.

7. Replace the top plate on the fermentor vessel. Secure it in place with the four retainer screws. Turn the screws until they are “finger tight.”

8. Replace the siphon and sparge tubes in the cover plate. Make sure that the ring on the lower end of the tube is centered directly below the impeller paddles. Position the siphon tube such that the lower orifice is centered and is as close to the bottom of the fermentor as possible. To the upper ends of these tubes (i.e., above the cover plate), attach short lengths (~6-8 in) of #25 Masterflex Neoprene rubber tubing. Cover the free ends of the tubes with blue Steri-Wrap paper. Then, cover with aluminum foil.

9. In addition to the sparge-, siphon- and sample tubes, the cover plate has three other stainless-steel addition ports used for making additions to the vessel during the fermentation (e.g., media additives, inocula, acid or base, etc.). To these ports, attach short lengths (~6-8 in) of Masterflex Neoprene rubber tubing with diameters that match those of the ports. Cover the free ends of the rubber tubes with blue Steri-Wrap paper. Then, cover with aluminum foil.

10. If the fermentor is to be sparged with air, oxygen or some other gas, the gas must be sterilized before entering the fermentor. Attach a filter to the sparge tube (#9 above). A suitable filter is the Whatman Hepa-Vent glass microfiber filter, 50 mm diameter, 0.3 µ pore size, GE-Healthcare Life Sciences (Cat. #6723-5000). Wrap the filter with blue Steri-Wrap paper. Then, wrap with aluminum foil. The filter will be sterilized during autoclaving. During set-up, the gas delivery tube is attached to the filter.

11. Replace the sample tube in the cover plate. If samples will be taken from the fermentor during the fermentation, attach a 50-ml centrifuge tube to the sampling port. Tighten the tube, then back off about ¼ turn. Close the valve on the port. This has a short, stainless-steel tube for applying a vacuum to remove samples from the fermentor. Attach a short (~2 in) piece of #25 Neoprene tubing to the tube. Insert a foam plug into the rubber tube. Then, cover the end of the tube with foil.

12. Cover the stirring gear of the stirring shaft and paddle assembly with a stainless-steel cap. Then, cover the cap with aluminum foil.

13. Prepare the medium to be used for the fermentation. Remove the rubber septum from the cover and, using a funnel, pour the medium into the fermentor. Replace the septum.

14. If necessary, attach the condenser to the cover plate (see Instruction Manual). As with the sparge tube, attach a filter to the open end of the condenser. Cover the filter with blue Steri-Wrap paper. Then, cover with aluminum foil.

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Operation: Setting the pH Probe

1. If the pH is to be monitored and/or controlled, attach the cable from the control module to the pH probe.

2. Calibrate the pH probe as follows:

a. On the main screen, press “Calibrate.” Then, select “pH.”

b. Immerse the probe in pH 7.0 buffer. When “Current Value” readout is ~7, click on the window below “Set Zero.” Type in 7.00 on keypad, then click “OK.” When “Current Value” stabilizes, click on “Set Zero.” This sets the probe to pH 7.0.

c. Rinse the probe with distilled water. Immerse the probe in pH 4.0 buffer. When “Current Value” readout is ~4, click on the window below “Set Span.” Type in 4.00 on keypad, then click “OK.” When “Current Value” stabilizes, click “Set Span” to set probe to pH 4.0.

d. Disconnect the pH cable and attach the end cap to the probe.

e. Insert the probe through the opening in the cover plate and tighten the retaining nut. Push the probe down until the probe tip is submerged in the medium.

Operation: Setting the Dissolved Oxygen Probe

1. If the dissolved oxygen (DO2) is to be monitored and/or controlled, the DO2 probe must be attached to the fermentor.

2. Service the DO2 probe as follows:

a. Unscrew the cylindrical membrane unit from the end of the probe. Clean the end of the probe with toothpaste. Then, rinse with distilled water.

b. Check to see if the membrane is intact. Remove the membrane from the probe and pour the electrolyte out.

c. Attach the membrane to a 10-mL syringe, then immerse it into a beaker of clean water. Press on the syringe plunger and check for air bubbles. If air bubbles exit the membrane, the membrane is perforated and must be discarded. Procure a new membrane and fill the interior about halfway with O2 electrolyte, then screw the new membrane on the end of the probe. If the old membrane is not perforated, fill the interior about halfway with O2 electrolyte, then screw the membrane on the end of the probe.

d. To check for functionality (i.e., to see if the probe is working), hook up the probe to the fermentor control module. On the Summary screen, press “DO2.” If the membrane is working, a positive numerical value will appear on the display. A negative number indicates a problem. If the membrane is an old one and was dry (i.e., contained no electrolyte solution), it may be necessary to wait 10-20 min to allow the probe to soak in the new electrolyte solution. If the probe is OK, then after a short while, a positive reading will appear on the display.

3. Calibrate the DO2 probe as follows:

a. Attach the probe by inserting the probe through the opening in the cover plate and tighten the retaining nut. Push the probe down until the probe tip is submerged in the medium. Attach the end cap to the probe.

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b. Setting the 0% and 100% O2 Set-Points are done after autoclaving, and when the fermentor and its contents have cooled. If any other media ingredients need to be added to the fermentor, they should be added prior to DO2 monitor calibration. Also, if the pH of the fermentor needs to be adjusted, this should be done prior to calibrating the DO2 probe. Be sure to set the agitation at ~200 rpm during the additions.

Operation: Autoclaving the Fermentor

1. Fasten screw clamps to the rubber tubes attached to sparge tube, the siphon tube, and (if necessary) any other tubes that go below the level of liquid in the fermentor. Tighten the clamps.

2. Check to be sure that the valve on the sample port is closed. If this is not done, the pressure build-up that occurs during the initial stages of the autoclaving process will force the media up through these tubes and out of the fermentor, where it will be “lost.”

3. It is advisable to sterilize the fermentor ~1 day in advance of the fermentation. Note: Prior to placing the fermentor in the autoclave, the vessel must be vented. This can be done by ensuring that the gas exhaustion pathway is clear and, as a backup, by loosening the inoculation port fitting slightly. If these steps are not taken, there is danger that the vessel may burst due to built-up pressure. Sterilize the fermentor as follows:

a. Place the fermentor into the autoclave.

b. Sterilize at 121ºC and 15 psi. Sterilization time will vary depending on the volume of media in the fermentor. For 2 L of media, a sterilization time of 90 min is adequate. For larger volumes, more time may be required.

c. After the sterilization process and when the autoclave has de-pressurized and cooled, remove the fermentor.

d. Re-attach the cable to the DO2 probe. This step is important, as the probe requires ~6 hrs to re-polarize. Otherwise, calibration of the probe and DO2 readings may be inaccurate.

Operation: Final Set-up

1. Hook up the condenser as follows:

a. Attach the Water-In and Water-Out lines to the condenser. Then, open the water supply.

b. Any live cells in the aerosol must be not be allowed to escape into the atmosphere. For this reason, attach a filter to the condenser outlet tube (#9, above). A suitable filter is the Whatman Hepa-Vent glass microfiber filter, 50 mm diameter, 0.3 µ pore size, GE-Healthcare Life Sciences (Cat. #6723-5000). Wrap the filter with blue Steri-Wrap paper, then with aluminum foil. As an alternative, omit the filter and instead cover the condenser outlet tube with Steri-Wrap and aluminum foil.

c. After the fermentation has started, attach the sterile outlet tube from the condenser to a piece of tubing leading to a sterile bottle with ~50-100 mL of bleach. If any aerosol with live cells passes through the condenser, they will be contained in the bottle with bleach (see “D” below).

d. Be sure that any unused delivery or sampling tubes are clamped off with a screw clamp. If not, any aerosol generated during sparging will “take the path of least resistance” and exit through one of these tubes rather than through the condenser.

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2. Control the temperature as follows:

a. Insert the temperature probe into the probe well on the fermentor lid.

b. Turn on the water supply to the control unit. The valve for the water supply is behind the control module and above the lab bench.

c. Attach the water lines from the control module to the fermentor.

d. On the control module screen, select “Temperature.” Enter the desired temperature and click “OK.”

e. Click “Auto.” This will allow water to circulate from the control module to the fermentor and raise or lower the temperature to the desired level. If temperature does not rise after control has been activated, check for vapor lock in the tubing.

3. Agitate the medium as follows:

a. Remove the stainless steel cap from the stirring gear.

b. Position the stirring motor on the stirring assembly, making sure that the gears of the motor and the shaft engage correctly.

c. Enter the desired agitation speed on the control module.

d. Click on “Auto” to start the stirring motor.

4. Set the 0% DO2 Set Point as follows:

a. Be sure that the cable is disconnected from the O2 probe.

b. On the control monitor, call up the Calibrate mode. Then, select “DO2.” Click the box below “Set Zero.”

c. On the resulting keypad, type in 0.00. Then, click “OK.”

d. On the DO2 calibrate screen, press “Set Zero.” This sets the probe at 0% DO2.

e. Connect the cable from the control module to the DO2 probe.

Note: When setting the zero, DO NOT leave the probe disconnected for more than one minute. Otherwise, after the cable is reconnected, the probe will need to re-polarize. This can take up to 6 hours. If the probe is calibrated to 100% and put into use without proper re-polarization, DO2 values obtained during the fermentation may be inaccurate. When setting the zero, be quick!

5. Set the 100% DO2 Set Point as follows:

a. Set the agitation to the level to be used during the fermentation (see page 41 of the CelliGen 310 Manual for agitation adjustment).

b. Connect the sparge tube to the sparge inlet on the fermentor. Be sure to remove the clamp on the sparge inlet tube.

c. On the control module summary screen, select “Air.” Then, select “O2 Enrich.”

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d. Open the O2 cylinder to feed O2 into the fermentor. This will sparge the medium so that it becomes saturated with O2. A convenient sparge rate is 1-L O2 per L of Initial Fermentor Volume (IFV) per min, or vvm. For 2 liters, set the O2 flow @ 2.0 L/min. This will control the flow of O2 to the fermentor.

e. From the “Synoptic” screen, select “Gas Flo”, then enter 2.0 L/min for the gas. Note that the sparge rate is a variable that needs to be determined in advance for the organism in question.

6. Fermentor Inoculation: Depending on the inoculum volume (~50-100 mL), it can be added to the fermentor using a large sterile syringe and a sterile large-bore needle. For volumes of several hundred mL, it is better to add the inoculum from a sterile bottle using a peristaltic pump. In either case, the inoculum is added through a sterilizable, self-sealing septum on the lid of the fermentor.

a. To Use a Syringe:

i. Aseptically, fill the syringe with the desired volume of inoculum.

ii. Sterilize the self-sealing septum on the lid of the fermentor with several mL of 70% ethanol.

iii. Insert the needle through the septum and push on the plunger to force the cells into the fermentor.

iv. Withdraw the needle.

v. Wash the septum again with alcohol.

b. To Use a Peristaltic Pump:

i. Procure a ~36” length of tubing (#24).

ii. Insert a 8-10” length stainless-steel tubing in one-end of the #24 tube.

iii. Insert the stainless-steel tubing through a rubber stopper on a screw-cap 0.5-1.0L bottle.

iv. Wrap foil around the cap.

v. Attach the other end of the #24 tubing to a wide-bore fermentor needle. Cover this with a screw-on metal “sleeve.”

vi. Autoclave the tubing and bottle assembly.

vii. After sterilization, aseptically transfer the desired volume of inoculum into the sterile bottle. Do this in the Bio-Safety Cabinet located in 1621 Food Sciences Building.

viii. Next, fasten a “loop” of the #24 tubing into the “head” of a peristaltic pump.

ix. Sterilize the septum on the fermentor lid with several mL of alcohol.

x. Uncover the needle and insert it through the septum.

xi. Turn on the pump to deliver the inoculum to the fermentor.

xii. After the inoculum has been added, turn off the pump, withdraw the needle and wash the septum again with alcohol.

xiii. Clamp off the tube on the addition port.

xiv. Disconnect the tube from the inoculum bottle.

xv. Cover the tube on the addition port with Steri-Wrap and aluminum foil.

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7. Foam Control: If the medium is foamy, add a few drops of sterile antifoam using a sterile needle/syringe assembly. Add the antifoam through the sterilizable self-sealing septum on the fermentor lid.

Operation: Harvesting the Fermentor

1. When the fermentation is complete, the fermentor contents are pumped from the fermentor into a collecting vessel (e.g., beaker, flask, etc.). Pumping is done with “house” air and the contents are pumped from the fermentor through a stainless-steel siphon tube that runs through the cover plate and reaches to nearly the bottom of the fermentor. DO NOT use oxygen to pump out the contents of the fermentor, as this can pose a risk of fire or explosion.

2. Attach a length (~24 in) of clean tubing to the upper end of the siphon tube.

3. Close/clamp off all other tubes leading to and from the fermentor.

4. With house air, pressurize the fermentor. Only slight pressure (~1-5 psi) is required for this step.

5. Collect the fermentor contents in a suitable vessel.

Clean-up Procedures

1. After the fermentor has been harvested, remove the septum on the fermentor lid.

2. Add sufficient distilled water (~1-2 L) to cover the ends of the DO2 and/or pH probes.

3. Clamp off all tubes that extend below the level of the liquid.

4. Autoclave the fermentor for ~2 hrs to kill all remaining microbes.

5. After autoclaving, allow the fermentor to cool.

6. Remove and clean all probes.

7. Remove the cover plate and dump out the water.

8. Clean the fermentor vessel inside and out with warm soapy water. Rinse with tap water, followed by a distilled water rinse.

9. Invert the vessel on several paper towels and allow sufficient time to dry.

10. Drain the contents of the mixing tank.

11. Clean the tank with warm soapy water. Then, rinse the tank with warm tap water, followed by distilled water.

12. Remove all clamps and rubber tubing from the cover plate.

Fully assembled Fermentor ~24-Hrs Post Inoculation

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13. Next, remove the sparge tube, sampling tube and siphon tube from the cover plate. Wash them with warm soapy water and, if necessary, pipe cleaners. Rinse with tap water, followed by a distilled water rinse.

14. Wash the top and underside of the cover plate with warm soapy water. Rinse with tap water, followed by a distilled water rinse.

15. Wash the impeller, using a brush to remove any cellular debris, etc., that may adhere to the paddles and shaft. Rinse with tap water, followed by a distilled water rinse.

16. Place the cover plate and all tubes on paper towels. Please allow sufficient time to these items to dry.

17. When dry, re-assemble the fermentor. This prevents individual components from being misplaced.

Machine Care and Maintenance

• The fermentor must be properly cleaned and inspected after each use.

• All inspections are performed by the Fermentation Facility manager.

• Report any problems to the Fermentation Facility manager.