Admit CO₂

Safety

Keep all PPE on, including cryogenic gloves: if the adapter leaks you tighten the cylinder joint further, under pressure. Keep the solenoid manual override closed (horizontal line pointing at 0 on the front of the solenoid).

The gas train was built and checked in CO₂ gas train, with the pin backed off.

1. Open the adapter’s pin to admit CO₂. The cylinder gauge reads about 60 bar when full. If the adapter leaks, tighten it further.
2. Screw the regulatorDrops the cylinder's pressure (about 60 bar when full) to a working pressure, about 1 bar here. Its flathead screw fully anti-clockwise is closed. in until its outlet gauge reads about 1 bar.
3. Remove the compression nut and ferruleThe ring inside a compression fitting. Tightening the nut squeezes it onto the tube to make a gas-tight seal. from the top of the needle valveA fine-adjustment valve on the solenoid's front port. It sets how fast CO₂ flows; turn clockwise to close., thread the nut onto the 4 mm polyurethane CO₂ tube, and push the tube fully onto the needle valve (dip it in hot water if it will not go). Refit the ferrule, and screw the nut down.
4. Cut the tube just long enough to run over the regulator and down to the vial’s CO₂ inlet (cut every other unit’s tube to the same length), and push a 1/8” hose barb to male luerA standard medical screw fitting. A male luer (the spigot) screws into a female luer (the socket). In this build, male ends mark outlets. lock adapter into the free end (hot water if needed).
5. Fit the male end of a 0.2 μm vent filterA 0.2 µm hydrophobic membrane on luer fittings. Gas passes; microbes and liquid do not, so each gas path stays sterile. to the female luer on the CO₂ inlet, and connect the tube’s luer to that filter.
6. Fit the female ends of two 0.2 μm vent filters to the male-to-male adapters on the two gas outlets (fitted in Ports), and cap any unused luer lock with a luer lock cap.
7. Route the solenoidAn electric valve on PWM channel 4 that opens to sparge. It must be 3-way venting (3/2), so the line vents when it closes. lead down behind the Pioreactor and through the pumps, and plug it into PWM channelOne of the HAT's five switched power outputs. In this build: 1 stirring, 2 media pump, 3 waste (product) pump, 4 CO₂ solenoid (relay), 5 heating. 4.

Notes

• CO₂ enters through the anodeThe positive electrode: a titanium tube coated with mixed metal oxide (IrO₂-Ta₂O₅). Oxygen forms on it, and CO₂ flows down its bore into the vial. and leaves through its open base. The gas rises past the anode surface and clears oxygen bubbles from it, with no separate spargeBubbling gas through the culture. Here, CO₂ from the cylinder flows down the anode on a timer set in the electroPioreactor job. tube.

Parts

• 1×CO₂ regulatorearlier step
• 1×CO₂ needle valveearlier step
• 1×Polyurethane CO₂ tube, 1 mpart
• 3×0.2 µm hydrophobic vent filterspart
• 1×1/8" barb to male luer lockpart
• 1×Silicone feed tube, tubular anodeearlier step
• 2×Male-to-male luer lock adapterearlier step
• 1×Luer lock cappart
• 1×Wiring, solenoidpart
• 1×MMO anode, 100 mm long, 6 mm OD, 4 mm ID, 70 mm active lengthearlier step
• 1×Crimp connector (TE Connectivity AMP 1-104479-0)earlier step
• 1×Crimp housing (TE Connectivity AMP 487526-1)earlier step

Tools

• 1×28 mm gas cylinder wrenchtool
• 1×Cryogenic glovestool
• 1×Eye/face protectiontool
• 1×Lab coattool
• 1×Multitool and/or needle nose plierstool

Check your work

1. With CO₂ admitted, where can you hear or feel gas escaping?

   • Nowhere along the train
   • At a threaded joint of the solenoid or needle valve
   • Where the 4 mm tube meets a barb or ferrule
   • Round the blanking plug in the solenoid
   • At the adapter on the cylinder
   Show the answer

   Answer: Nowhere along the train

   • If you see At a threaded joint of the solenoid or needle valve: Every threaded joint without an o-ring seat needs Loctite 577An anaerobic thread sealant: it cures only without air, inside a tightened thread. Fixtures in 10 to 60 minutes at 22 °C; full pressure after 24 hours.. It fixtures in 10 to 60 minutes at 22 °C and reaches full pressure rating after 24 hours.
   • If you see Where the 4 mm tube meets a barb or ferrule: Soften the tube end in hot water and reseat it. The 1/8" barb must grip the 4 mm tube, or the joint leaks under pressure.
   • If you see Round the blanking plug in the solenoid: It seals on its o-ring, not on sealant. Check the o-ring is present, and tighten with the wrench.
   • If you see At the adapter on the cylinder: Tighten the adapter further with the gas cylinder wrench, wearing the cryogenic gloves.

2. Which PWM channelOne of the HAT's five switched power outputs. In this build: 1 stirring, 2 media pump, 3 waste (product) pump, 4 CO₂ solenoid (relay), 5 heating. is the solenoidAn electric valve on PWM channel 4 that opens to sparge. It must be 3-way venting (3/2), so the line vents when it closes. connector plugged into?

   • PWM 3
   • PWM 4
   • PWM 2
   Show the answer

   Answer: PWM 4

   • If you see PWM 3: Move it to PWM channel 4: the plugin maps that channel to the relay. PWM 3 is the product pump's.
   • If you see PWM 2: Move it to PWM channel 4: the plugin maps that channel to the relay. PWM 2 is the media pump's.

≈ 0.02 g CO₂e per view