BioFlow 3000 Fermentor Notes

Rinse vessel out. Use warm water and soap if necessary; no abrasives.
Measure and mark out final volume on vessel wall. Pour in prepared media (4 liters minimum; use 6 liters for RNA studies). Add antifoam (1 ml antifoam/10 L media for Sigma A5551):
- 120 g tryptone
- 60 g yeast extract
- 60 g NaCl
- 0.6 ml antifoam
  ->up to 6 liter mark with H₂O
Calibrate pH meter with pH 4, pH 7 buffers.
Insert into headplate and cap: pH and DO electrodes. Insert level probe. Make sure sampling vial port is closed; harvest port is clamped off. Autoclave fully assembled vessel in big autoclave (WAB 573, Media Prep) in a tray of water using Liquid cycle, 35 min, T=250^oF. Make sure vessel is not pressure sealed or it will EXPLODE in autoclave: loosen clamps on antifoam and/or acid addition ports.
When vessel is cool enough to remove from autoclave (1 hr from opening door), take out and place on console to fully cool overnight. Attach polarizer to DO sensor.
Start agitation, temperature control, and air flow. Bring media to incubation temp (37^o), 30 min-1 hr.
Calibrate DO sensor: 100% to fully oxygenated media. Remove sensor connection for 0%.
Rezero pH electrode to pH 7.
Add antibiotic if necessary.

Notes:

If antifoam is being added by a control loop, use the level probe. Console control loop is Level 1, 2, or 3. Set Control to Harvest (so if media level hits probe, pump will turn ON to pump in antifoam).
If using pump to harvest culture, we have to develop our own protocol.

pH Control Loop (Use either base or acid; not both)

Set pH Control to P-I-D.
If using base to control pH, use lowest feed pump (Feed 5) so any leaks do not drip to lower pumps. Set Feed 5 Control to Base.
For Set duty-cycle values:
- 125 = maximum, always on
- 100 = 10 sec on, 3 sec off
- 50 = 6 sec on, 6 sec off
Note: Pump velocity is constant, so flow rate is varied by tubing diameter.