Ports

The cap has eight ports. The electrodes already fill two, this step puts needles through four, and the septumThe 2 mm silicone disc in the vial cap. It seals the vial mouth, both electrodes and every needle port, and self-heals needle holes. keeps the last two closed:

• 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 CO₂ In (6 mm)
• CathodeThe negative electrode: a stainless steel rod. Hydrogen forms on it, so it shows about twice the bubbles of the anode. It must never be wired as the anode. (6 mm)
• Media In
• Media Out
• Gas Out
• Gas Out – safety
• Inoculation (the large port): a syringe goes straight through the septum, with no tube and no pinch slider
• Spare: left unpierced, so the septum seals it

1. Push a female luerA standard medical screw fitting. A male luer (the spigot) screws into a female luer (the socket). In this build, male ends mark outlets. onto the media pump’s inlet tubing. It is a tight fit (1 mm bore).

2. Push the 80 mm needle (Media Out) down through the septum to the liquid level you want: it sets the level.

3. Push the 75 mm needle (Media In) straight through the septum.

4. After each needle goes through, pull it back out, clear any silicone plug, and reinsert it through the same hole.

   Tip

   Light visible through the needle means it is clear. Otherwise blow it clear with a syringe.

5. Push the two bent needles (Gas Out and Gas Out – safety) through the septum, above the liquid level.

6. Fit a male-to-male luer adapter to each of the two gas outlet needles, so outlets are marked as outlets.

7. Fill the vial with DI waterDeionised water: water with its dissolved ions removed. Used here to set the vial level by weight, 1 g per ml. via the pumps, and weigh it against the dry empty vial.

8. Move the Media Out needle up or down until the vial holds 30 ml and Media Out just maintains that level while the pumps run.

9. Measure the electrode immersion depths, adjust them to the standard if needed, and record each insertion depth.

10. Check the Media Out needle is at the water level, every other needle is higher, and none is plugged with silicone.

    Bent needles

    You cannot look through a bent needle. Blow gas or air through it at a steady pressure and compare the flow with a clear needle.

11. Set up the Pioreactor in turbidostat mode.

Parts

• 4×Stainless steel needle portpart
• 2×Male-to-male luer lock adapterpart
• 1×MMO anode, 100 mm long, 6 mm OD, 4 mm ID, 70 mm active lengthearlier step
• 1×Stainless steel cathode, 100 mm long, 6 mm diameterearlier step
• 1×Vial cap and electrode holderearlier step
• 1×Silicone septum, 2 mm thick, 60 Shore Aearlier step
• 1×Pioreactor 40 ml glass vialearlier step

Tools

• 1×Analytical balancetool
• 1×Vernier calliperstool

Check your work

1. Which of the cap's eight ports have nothing through them?

   • Inoculation only
   • The spare only
   • Inoculation and the spare
   • Inoculation, the spare and a gas outlet
   Show the answer

   Answer: Inoculation and the spare

   • If you see Inoculation only: A needle went into the spare port. Pull it out (the septumThe 2 mm silicone disc in the vial cap. It seals the vial mouth, both electrodes and every needle port, and self-heals needle holes. self-heals the needle track and keeps the port sealed) and put it through the port it belongs to.
   • If you see The spare only: A needle went into the inoculation port. Pull it out: the inoculation syringe goes straight through the septum there. Put the needle through the port it belongs to.
   • If you see Inoculation, the spare and a gas outlet: Push the second bent needle through as Gas Out – safety. Short of needles? Each Pioreactor ships with four, and the BoM adds one per unit: check Count what you received in Before you start, and use the Missing something? line under it.

2. Where do the needle tips sit relative to the water?

   • Media Out at the surface, all the others above it
   • Media In under the surface, the others above it
   • A bent gas needle at or under the surface
   Show the answer

   Answer: Media Out at the surface, all the others above it

   • If you see Media In under the surface, the others above it: Pull Media In up above the water: only Media Out sits at the water level.
   • If you see A bent gas needle at or under the surface: Pull it up above the liquid level: only Media Out sits at the water level.

3. Filled with DI waterDeionised water: water with its dissolved ions removed. Used here to set the vial level by weight, 1 g per ml. via the pumps and weighed against the dry empty vial, what does the vial hold while the pumps run?

   • More than 30 ml
   • 30 ml, holding steady
   • Less than 30 ml
   Show the answer

   Answer: 30 ml, holding steady

   • If you see More than 30 ml: Push the Media Out needle a little further down, run the pumps and re-weigh until it holds 30 ml.
   • If you see Less than 30 ml: Pull the Media Out needle up a little, run the pumps and re-weigh until it holds 30 ml.

4. What have you recorded for the electrodes in this step?

   • Each insertion depth, before setting the standard immersion
   • Nothing yet for either electrode
   • Each insertion depth, after setting the standard immersion
   Show the answer

   Answer: Each insertion depth, after setting the standard immersion

   • If you see Each insertion depth, before setting the standard immersion: Adjust them to the standard immersion depth, then record the new insertion depths.
   • If you see Nothing yet for either electrode: Measure the immersion depths, adjust them to the standard if needed, and record each insertion depth.

≈ 0.02 g CO₂e per view