1. Gather all bioreactor vessel parts, including the pH probe and accessories (calibrating buffers and aqueous saturated potassium chloride solution for filling the pH probe) and the DO probe.

2. Clean all bioreactor vessel parts (except pH and DO probes) with a soft cloth (a soft brush may be used on difficult to clean parts of the bioreactor vessel), warm water and liquinox. Rinse with warm water and several changes of deionized water.

3. Clean the pH and DO probes using a soft cloth and deionized water. Be careful not to harm the sensory tips of both of these probes.

Set-Up Procedure

A. Assembling the Vessel

1. Attach impellers to the shaft. Lower impeller should be at the base of the shaft, and the top impeller should be one impeller diameter (6 cm) from the base of the lower impeller. (Ideally the impellers will not need to be removed after each run in which case this step can be omitted.)

   For the following assembly instructions, all fittings are hand-tightened. Use no tools.

2. Insert all probes/tubes that attach from the under side of the head plate, including the sparger tube and harvest tube. Lubricate seals by adding a small drop of glycerol to the o-rings before inserting anything. 

   * If not harvesting, place a steel plug into the harvest port.

3. Insert all probes/tubes that attach from the top side of the head plate, including thermowell, sampler assembly, concenser, and foam probe.
4. Place baffle assembly into the glass vessel such that the 2nd baffle (counting around the ring counter clockwise) is centered between the addition port directly below the New Brunswick logo and the port to its left.
5. Replace the fermenter vessel onto the BioFlo stand.
6. Pour in media, make sure that both impellers are submerged.
7. Place the head plate onto the vessel and lock it to the clamping ring by tightening the thumb screws. ( Add a few drops of glycerol to the o-ring on the underside of the head plate to insure a good seal between the head plate and the glass vessel, and between the flat o-ring and stainless steel head plate ring.)
8. Add 9 ml of Antifoam-A through the inoculation port.
9. Insert the temperature probe (RTD) with a few drops of glycerol into the thermowell and plug into the BioFlo 3000.
10. Place steel blank into condenser port with a few drops of glycerol and gently tighten.
11. Attach the acid/base silicone tubing (I.D. 1/32) to one of the addition ports. Bend tubing in half and secure with a twist tie such that nothing can escape through the tubing.
12. Wrap cotton and aluminum around the 2nd addition port.
13. Wrap cotton and aluminum foil around the harvest tube.
14. Place 1/4 I.D. silicone tubing on the top of the sparger tube and connect the air filter such that there is tubing attached to both sides. Wrap cotton and aluminum foil around the exposed tubing end.
15. Loosen the inoculation port to allow ventilation.
16. Loosen the sampling tube, and remove the rubber bulb, make sure that there is an ample amount of glass wool in tube where the rubber bulb connects to the sampling assembly.
17. Make sure that the sampling valve is closed.
18. Connect the water out line to the vessel heat exchanger (the vessel base).
19. Connect the water in lines to the vessel heat exchanger.
20. TURN ON WATER (under the counter)

    The pressure gauge should read 15 psig.

21. TURN ON AIR

    The pressure gauge should not exceed 10 psig, 5 psig is safe.

22. TURN ON BioFlo 3000.

1. Check the probe for any trapped air bubbles, tap gently at a 45 degree angle to remove bubbles.
2. Check electrolyte levels within the probe, they should be around 1 cm below the each of the filling ports.
3. Remove the rubber plugs.
4. Attach one end of the pH cable to the pH probe and the other end of the pH cable to Bioflo console.
5. Go to <Calibration> screen
6. Immerse pH electrode into a pH 7 buffer solution.
   Allow a few minutes for the electrode to equilibrate. 
7. Set function column to zero by pressing alter and press enter.
8. Enter seven (7.0) under the zero column and press enter.
9. Rinse electrode thoroughly with de-ionized water.
10. Immerse pH electrode into a pH 4 buffer solution.
    Allow a few minutes for the electrode to equilibrate.
11. Set function column to span and press enter.
12. Enter four (4.0) under the span column and press enter.
13. Rinse electrode with de-ionized water
14. Repeat calibration.
15. Apply a small amount of glycerol to the probe prior to inserting into the vessel.
16. Disconnect the cable, and replace shorting caps and rubber plugs prior to sterilization. Rubber bands, located on the pH probe, should be placed over the rubber plugs.

1. Remove protective cap from the electrode end.
2. Attach adapter to head plate and tighten with a wrench.
3. Carefully insert the probe into the adapter.
4. The shorting plug should be installed prior to sterilization.

1. Turn off the power.
2. Turn off the water.
3. Turn off the air.
4. Disconnect water out lines to heat exchanger.
5. Disconnect water in lines to heat exchanger.
6. Remove RTD from the thermowell.
7. Autoclave entire assembly at 121 degrees Celsius at 15 psig for 25 minutes.

1. Place the BioFlo vessel onto the console.
2. Connect the water out line to the vessel heat exchanger (the vessel base).
3. Connect the water in lines to the heat exchanger.
4. Connect the water out line to the condensor. (top)
5. Connect the water in line to the condensor. (bottom)
6. Add glycerol to thermowell and insert RTD.
7. TURN ON AIR
   Approximately 5 psig, do not exceed 10 psig. 
8. TURN ON WATER (under the counter) Make sure that the water pressure is between 15 and 20 psig.
9. Connect the air line to the sparger port.
10. Turn on power switch.
11. Select Fermentation mode (#2)
12. Go to <Master> screen and set the temperature control mode to PRIME for one minute.
13. After a minute set the desired working temperature (30 degrees C) and the control mode to PID.
14. Remove the rubber plugs and shorting cap from the pH probe and connect the pH cable.
15. Remove shorting cap from DO probe and connect DO cable.
16. Place motor onto the top of the head plate and plug into the console.

1. Go to the <Gases> screen and set the mode to manual, DO NOT PRESS ENTER.
2. Remove the foil and place exhaust tubing, and filter such that is collects into a beaker that is placed beside the BioFlo console.
3. Press enter.

1. Place the agitation loop into PID control and set the set point to 100 rpm.
2. Attach the ammonium hydroxide through the peristaltic pump and to the addition port that already has the tubing attached to it. (To thread the tubing into the peristaltic pump use the diagram of the sampling procedure in the Bioflo manual and thread the tube in the opposite direction to bring solutions into the vessel.)
3. Set the pH loop to PID control and set the set point to 5.8.
4. Set the feed pump 1 control loop to BASE, and the set point to 100. Do not press enter.

H. Calibration of the Dissolved Oxygen Probe

NOTE: Probe can not be calibrated until the desired working temperature has been reached.

1. Go to <Calibration> screen, arrow to Function column for DO.
2. Unplug DO cable from the DO probe.
3. Set Function column to zero and press enter.
4. Arrow to zero column and enter zero (0.0) and press enter.
5. Reattach cable to DO probe.
6. Go to <Master> screen and set Agitation to 1000 rpm.
7. Go to <Gases> screen and set the mode to manual.
8. Allow ten to thirty minutes for the vessel to equilibrate.
9. Go to <Calibration> screen, arrow to Function column for DO.
10. Set Function column to Span and press enter.
11. Arrow to Span column and enter 100.0 and press enter.

I. Three Possible Control Modes for Dissolved Oxygen

Controlling by agitation only

1. Set agitation and dissolved oxygen to PID control.
2. Set minimum rpm for agitation.
3. Change agitation to DO control mode.
4. Set maximum rpm for agitation.

Controlling by % of oxygen sparged

1. Set DO control mode to PID.
2. Go to <Gases> screen and set mode to DO.

Controlling by Agitation and % of oxygen

* Will adjust agitation until maximum/minimum is reached and then will switch to control by % oxygen.

1. Go to screen. Set dissolved oxygen and agitation control modes to PID.
2. Set minimum rpm for agitation by changing the set point to desired minimum (50 rpm).
3. Set agitation mode to DO.
4. Set maximum rpm for agitation by changing the set point to desired maximum (300 rpm).
5. Go to <Gases> screen and set mode to AgO2DO mode.
6. Go to screen and alter to air monitor (the air flow rate should be around 1.5 to 2 L/min), adjust the air pressure accordingly.

J. Use of Anti-foam Sensor

NOTE: This feature will not be used in the fermentation of Pichia.

1. Connect cable to level 1 hook up.
2. Connect the black cable to the Anti-foam sensor.
3. Connect the red cable to the ground pin located on the head plate.
4. Place Anti-foam sensor such that the tip of the sensor lies at the desired level.
5. Go to <Master> screen and alter the loop name to read Feed 1 and press enter.
6. Set the control mode to LV1 (level 1).
7. Set the set point to the desired percentage that you would like to add the anit-foaming agent once foaming reaches the level of the Anti-foam sensor.
8. Alter loop name to read Level 1 and press enter.
9. Set the control mode to Add.

Fermentation Procedure

At this point we are ready to do the final preparation before inoculation. Our fermentation run is going to be monitored and controlled by a computer using the AFS (Advanced Fermentation Software) software package (New Brunswick Scientific). Once the computer has been turned on and set up, we will no longer be able to change the set point at the console. The only use of the console control panel at this time is to set the actual control modes on/off.

A. Introduction to AFS Software

1. Turn on computer.
2. At c:\ prompt type cd\afs and press <enter>.
3. At c:\AFS> prompt type afs and press <enter>.

At this time the main menu will appear on the screen. Important: Once the computer has been turned on and the afs program set up you can no longer change the set points at the Bioflow console. All changes must be made at the computer.

B. Setting Up the Program

1. While set-up is highlighted press enter.
2. Type in com port 2.
3. Set MD# (multidrop number) to 0, Active to Y (yes). You can press the enter button to move around the screen.
4. Press <ctrl> <enter>.

C. Setting Up a Data Log File

1. Set parameter by using either the arrow keys or the tab keys to move around. For each parameter you wish to log (record data) you need to type in Y (yes) under unit #1. Press <ctrll><enter> to save.
2. Highlight unit #1 and press <enter>.
3. Enter log filespec in this form: filename.log
4. Enter log identifier: type prn
5. Set log interval in minutes, minimum log time is 1 minute, we will use 15 min.
6. Set logger active to Y (yes).
7. Press <ctrl> <enter>.

D. Menu Items in the Monitor Screen

1. Alarm: This allows you to set minimum and maximum values such that if at any time during your run your current values reach its max. or min. an alarm will sound.
2. Set Point: this allows you to change the current set points of the different parameters.
3. SupvCtrl: Allows you turn on or off control equations that are user written.
4. UserEq: Allows you to write user equations.
5. EFT: Allows you to reset your elapsed fermentation time.
6. Trend: Allows you to construct and display a graph of selected values of your parameters vs. time and view the changes during the run.
7. Return: Takes you back to the Main menu.

E. Inoculation Procedure

1. Allow the fermenter time to reach all of its set points.
2. Add 13 mls of the trace salts solution through the inoculation port using sterile technique.
3. Clear the EFT.
4. Photodocument the inoculum culture.
5. Add the inoculum through the inoculation port using sterile technique.

F. Monitored Parameters

Once the fermenter has been inoculated the following parameters should be monitored no less than twice a day. Each time a sample should be taken.

1. Immediately measure and record the OD.
2. Using the LC-100 quantitate the HSA concentration.
3. Spot slide and take a picture, date and save in appropriate file. Check for contamination.
4. Check the amount of air/oxygen left in tanks and hook up new tank if less than 500 psig of air/oxygen left.
5. Check each of the set points and make sure that the computer is properly adjusting the parameters.
6. Check for foaming, add more anti foam A if necessary.

Shut-Down Procedure

1. Turn all the control modes on the console to off.
2. Turn off the Bioflo 3000.
3. Remove the motor and wipe down with a sponge using a bleach/water solution.
4. Remove all probes and clean thoroughly. DO and pH probes should be stored upright. The pH probe should have its tip immersed in saturated KCl.
5. Remove all plugs, swage nuts, and o-rings from the head plate and clean thoroughly.
6. Remove the head plate from the vessel.
7. Detach all the probes that connect form the underside and clean thoroughly.
8. Clean head plate and impellers with soft brush or sponge.
9. Pour excess media via funnel into flasks and then autoclave.
10. Detach glass vessel and clean carefully with a sponge (glass is very brittle).
11. Clean heat-exchanger base with a soft brush, removing o-ring.
12. Rinse all parts with DI water and let air dry.
13. Wipe down the Bioflo console with bleach/water solution.

TOP
Bio-Link.org
Web: www.Bio-Link.org
Email: info@Bio-Link.org
NSF Award #9850325