Lidstrom: Bioflo 310 Setup

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System Info

• New Brunswick BioFlo 310: Benchtop Fermentor/Bioreactor
• Shimadzu GC
• Mass flow controllers
• Optical Dissolved Oxygen Probe
• pH probe (orange topped one from Broadley James)

Preparing the Bioreactor

Calibrate the pH probe

1. Turn on the controller using the big green switch on the front
   • It should launch to the summary screen
   • Use the touch screen to navigate to the Calib tab
   • Check pH-1
2. Pull out the probe
3. Rinse with DI water
4. Attach the probe to the pH line (The port is labeled on the back of the controller unit)
5. Calibrate to pH 7
   • Click the blank under the Set Zero
   • Input 7.0
   • Wait until the raw value stabilizes
   • Press set zero
6. Rinse the probe and set in water while you pull out the next solution
7. Calibrate to pH 10.
   • Click the blank under the Set Span
   • Input 10.0
   • Wait until the raw value stabilizes
   • Press set span
8. Rinse the pH probe.
9. Disconnect the probe and keep it moist (Alexey puts it briefly in a wet paper towel).

Set up the vessel

Open up the bioreactor vessel a. Remove all probes first and set them aside to prevent damage because they are expensive the breakable i. pH probe ii. DO probe iii. Level probe (unscrew surrounding bolt to make it easier to move in and out. iv. Sampling port v. Condenser (undo the clamp while holding the unit) b. Unscrew the four bolt holding the top of the vessel on. 3. Rinse the vessel and its components with MilliQ water a. Run water through the lines (do before and after a run)\ i. Sparging line ii. Inlet line and associated tubing iii. pH/baseline iv. Outflow line v. Sample port vi. The condenser column b. Rinse other parts i. Remove any biomass that you see ii. Scrub as needed. No soap. iii. Rest the bioreactor top rest on the motor side not on the metal tube side. (Failing to do so could damage the metal tubes.) 4. Partially assemble the bioreactor. a. Turn the bottom of the unit so that the water inlet and outlet are on the left side. b. Insert the baffle so that the opening faces ~6:30-8 o'clock." c. Put the lid on and make sure that the pH and DO probes will clear the baffles. d. Pull the lid back off and make the media. 5. Make NMS2 to fill the bioreactor a. 1 L base media with 4 ingredients. i. 0.2g MgSO4 *7H2O ii. 0.016 CaCl2*2H2O iii. 1g KNO3 iv. 7.5g NaCl v. Add 1L MilliQ Water with the graduated cylinder b. Alexey's best practice is to pull all the containers out to start and then replace them when that ingredient has been added. 6. Finish reassembling the bioreactor for autoclaving (you can also just assemble and skip the autoclaving step if you have pre-made medium and don't care to be sterile.) a. Keep the condenser separate. The o-ring should stay on the top of the bioreactor, not with the condenser. b. Put the lid on c. Check the fit of the baffles and probes. d. If everything is good, remove the probes. e. Screw the lid on by tightening the 4 bolts.

f. Put in the sampling port with the valve closed. g. Push in the level probe h. Put in the dissolved oxygen probe (loosen the surround bolt if need, but tighten after the probe is in). i. Screw in the pH probe.

7.

8. 9. Autoclaving the Bioreactor a. Foil open ports and line ends i. Clamp 2 out of 3 lines on the triport. (the base line isn't clamped) ii. Foil the metal tube and exposed ends of the sampling port iii. Foil pH probe and DO probe tops. iv. Foil 3 line ends on the tri port v. Foil condensing column input vi. Foil end of outlet line. Clamp outlet line vii. Foil the top of the motor or cover it with the rubber cap. viii. Foil the draw tubes for reservoir media container and tube ends ix. Foil the end points on the condensing column. x. Foil the end points on the sampling port. Make sure the red valve is closed (should be perpendicular to the assembly arm. b. Tray with Bioreactor. Make sure there are vents (just aluminum foil covered openings that steam can escape from.

c. d. Tray with condenser and stopper for inlet media bottle.

e. Autoclave on Cycle 2. f. Let cool. g. Once it's cooled a bit, you can hook up the temperature sensor/water flow for control to help cool the unit. 10. Finishing the medium and inoculating (on the bench across from the bioreactor controller. Use the flame.) a. Add trace to 1x through the condenser port. 2 mL/L media b. Add 20x phosphate. 20 mL/L media c. Add 1M carbonate. 25 mL/L media (reduced by half from normal recipe - this is not added to inlet media for chemostat mode) d. Use a pink needle and 60 mL syringe to inoculate 50 mL of overnight culture into the bioreactor. 11. Add the condensing column pointed away from you and slightly to the left. 12. Move bioreactor and turn off flame a. Put the bioreactor vessel in place for the run 13. Connect the water for the heat exchanger.

a. Connect the water outline to the base (red) b. Connect the water in line to the base (blue) c. Turn on the water by the hood. d. Make sure the pressure doesn't exceed 10 psi. 14. Connect the condenser and the gas outlet. a. Connect the water outline. b. Connect the water in line. c. Connect the gas out line and make sure the line is unclamped. 15. Connect the gas in line a. Plug in the mass flow controller. Should be set to 100 sccm. i. We have two gas flow controllers: one does high flow rates, the other does 1-10 sccm. ii. For the low flow one, gas mix 2 is 20% Ch4, 5% O2, 75% N2. iii. If you're using the external mass flow controllers and need to move the input line from the back of the system controller, plug in the house air to prevent errors. Use clamps while moving gas lines if they're on. iv. If you're using the system controller's gas regulator, set the total gas flow to auto and set the air (input 1) to 100%. b. Connect the gas in line (one with the big filter) c. Open the gas tank that you're using all the way. d. Check the level of gas in the tank. Even when the meter says zero there could still be a couple days' worth. e. With the agitation off, check for bubbling to make sure the bioreactor is getting gas. 16. Insert the temperature probe into its well. 17. Put on the motor. 18. On the summary screen, hit agitation ant turn it to auto (Alexey uses 750 for the NSF fermentation stuff and 1000 for the ARPA-E stuff). 19. On the summary screen, hit temperature and turn it to auto (T= 30 deg C).

20. Prep 2 M NaOH for base line 21. Plug in the pH probe to the pH-1 line. 22. Connect the baseline a. In the software, navigate to the "Pumps" tab in the controller. b. Change the pump identity to "None." c. Set to 100% and hit on to cause the pump to spin without a line in it. This is to note which way the pump is pushing so you can insert the tube in the right orientation. d. Thread the line from the base bottle through the pump so that the pump is moving liquid out of the bottle. e. Pump the end of the line in a vial and turn the pump on until liquid flows out the end. f. Make sure there are no clamps in the middle of the line. g. Clamp the end of the line. h. Attach the clamped baseline to the base line in the triport. i. Unclamp the baseline on the bioreactor side and the base bottle side. j. Set the pump to 5% and continue priming the line just into the skinnier diameter tubing. k. Once primed, change the pump 3 identity to base. 23. Go to the Summary screen and turn pH control to auto (pH set point = 8.8) 24. Prep 3 mL syringe with antifoam a. Stuktol J669R, antifoam, store at 4 deg C. Food grade. b. Put down foil or paper towel because it's messy. c. Remove the needle and suck up a full syringe worth. Cap with a new pink needle and prime. 25. Start the antifoam line a. Place the syringe in the syringe pump and set the rate to 20 uL/hr b. Insert the needle into the rubber septum in the bioreactor top at a right angle. c. Hit start on the syringe pump. 26. Set up the dissolved oxygen probe.

a. Connect the wireless card unit to the top of the probe. It should screw in. b. Connect the power cable. c. Open the Hamilton software on the computer. d. Right click on the Wireless port --> Additional functions --> Scan for devices --> DO <xxxxxx> should appear e. Right click on the probe and choose "Go Online." f. Navigate to the data log tab.

g. Choose your recording frequency. h. Set your file name. i. Hit apply to start recording data. 27. Take an initial sample and log the OD600 in the run excel file. (1:10 dilution for OD measurements - 900 uL media +100 uL sample) a. Removed ~ half the sampling vial and discard to clear line before taking the sample you measure.

28. Run in batch mode for ~24 then switch to chemostat. Switching to chemostat mode 1. Prep extra media for the inlet a. Add the trace and phosphate b. Unwrap the draw tubes attached to the stopper and place in the extra media vessel. 2. Attach the inlet line a. Thread the tubing through the pump. Use the buttons to choose a direction. The threading is a bit finicky. In general the tubing should lay flat along the outer surface of the pump circle. Not too much tension, but not too much slack. Should give a continuous pull.

Set the pump to 30 (the highest setting to prime the line). Then drop it to whatever number your calculations give (for FM82, we're using 5.21 for ~10 hour doubling time.) a. Connect the line to the correct housing on the triport valve. 3. Set the level probe a. Connect the level probe with a line from Level 1. b. Level the ground probe on the side of the base so it's always touching metal but out of the way. c. With the agitation at the desired level, pull the level probe up then push it down until the pump flicks on and off. d. Note an initial volume estimate in the excel file by turning off the agitation for a minute. 4. Attach the outlet line a. Connect the outlet line to the longer tubing that goes to a new container. Make sure it's empty because we will want to measure this volume. Anytime you pull out liquid, it goes back in this container. If it doesn’t, make a notation of how much in the excel file.

The cells should look good and happy when you spin them down.

• For growing cells, 1-2 doublings suggested, 0.6~0.8 OD600, for my barely utilizing cells, Yanfen suggest 50-100 mL

1. Label 50 mL falcon tubes
2. Setup Filtration Equipment
   • Filtration setup with vacuum flask (see pictures below)
   • Filters, Nylaflow, Nylon Membrane Filter 0.2 um, 90 mm, P/N 66603 [Pall Life Sciences]
   • Flat-tipped tweezers
   • Liquid nitrogen in Dewar