CO2 sparger drawback

I could do with a quick sanity check, so I’m tagging you @“Trusted”#g6 as I have 2h to place an order that may fix things in time for tomorrow’s video.

I set up an [FZONE CO2 Regulator and Solenoid](https://amzn.eu/d/gSnMfxg) on [an adaptor](https://amzn.eu/d/1eZLJ7n) on a [Sodastream cylinder](https://amzn.eu/d/1dZqKsi) then bodged a Luer lock fitting so that it could adapt the gas line down from what I assume is 3/16" to the 1/16" silicone tubing that we use with Pioreactor lids:

![Imgur](upload://2PrIFBoKDxbLxwtUMX8rkjmJ6fU.jpeg)

that's what I was upto at the start of yesterday's Journal Club.

When I returned this morning, the FZONE's bubble counter was full of Pioreactor culture:

![Imgur](upload://5ACK7mccoomNepC2Xsk6ff6ZRSE.jpeg)

My assumption is that the system happily pressurises and small leaks around the luer bodge aren't an issue so it happily sparges CO2. Then when the gas is turned off, the pressure equilibrates with a certain positive pressure in the CO2 apparatus. The luer bodge then allows CO2 to gradually escape bringing the CO2 apparatus pressure down to ambient, given that this is a lower pressure than the previous equilibrium, it draws my culture back up the sparger tube and into the bubble counter.

Does that sound most likely, or can you think of other reasons? I tried again with the Pioreactor vial cap on & the same thing happened, so I'm pretty confident we have ambient pressure in the Pioreactor.

This clearly wouldn't happen if I used @"CamDavidsonPilon"#5's recommended pinch valve, but I imagine if I can get the system CO2-tight, we should be OK..?

So I'm thinking about getting some [aquarium tubing](https://amzn.eu/d/fFg5mFa) (as recommended by Amazon next to the FZONE hence I assume it will fit) and some a [3/16" Male luer connector](https://amzn.eu/d/h5haiHQ) so I can connect into pioreactor sparse tube.

I’m not sure if this will work as there will be quite a volume of pressurised CO2. Presumably any leak in that system will resulting in a gradual decrease in pressure & drain the Pioreactor again.

All thoughts welcome, but I should order soon...

@“Martin”#p93 Yeah that sounds like the most likely scenario to me. I’d say yeah first work on getting it CO2 tight and then if that doesn’t work, a valve may be needed. Ideally you’d have it both be CO2 tight and have a valve as safety measure but for the time being hopefully using the correct luer connectors will help.

Instead of trying to keep the system CO2-tight, I’m wondering if you could modify this baffle design to create a sparger that partly sits above the media so that it creates an air gap and prevents backflow. You’ll lose some CO2 into the headspace, but maybe that’s okay?

Could putting the cylinder at a different height compared to the bioreactor help to prevent backflow?

Alternatively, you could install a [check valve](https://www.co2supermarket.co.uk/co2-check-valves-gc24.html).

Brilliant, thanks guys. I added 10 v. cheap check valves to my order.

UPDATE - I’ve just read that these check valves aren’t suitable for CO2, I imagine that’s because of the pressure, and note that they look decidedly similar to the £1.99 each “Plastic CO2 Check Valve” seen in Gerrit’s link - it’s too late to get anything else delivered tomorrow, so I’ll give one a go - if nothing else I like the fact that you can see what’s happening with them

@"RobJ"#8 I'm not sure that you'd be able to fit a baffle, 2 electrodes, 2 gas and 2 liquid lines all into one functioning cap that can still be attached to the vial with the platinum/titanium mesh attached, but feel free to prove me wrong...

I'm also not quite sure that it would do everything required in terms of purging oxygen (what do you think @"NarcisP"#9 ?)

Conversely the amount of CO2 I put through my beleaguered spirulina culture must have pushed pH way below anything that the few remaining survivors could cope with... @"Rafik"#7 is excessive carbonic acid an issue that you've come across?

@“Gerrit”#p95 I can’t quite visualise how the baffle design would be modified, any chance you could do a small sketch?

A check valve would certainly be a nice quick fix as well though

@“RobJ”#p97 What I’m imagining is a sparger that’s basically a combo of those two baffle designs on Printables. So you’d have a gas line going in, and sparging happens through all the tiny holes. Some of the holes will be in the headspace of the bioreactor, creating an air gap to prevent backflow.

Of course, It could also be the case that all the CO2 goes into the air gap instead of into the water. 🤷

@“Gerrit”#p98 yea my bet’s on the latter - IMHO you’d either need a jet of CO2 penetrating the surface to get any bubbles making it under the water, or the tiny holes above the surface would need to be so tiny that the CO2 flow entering the sparger exceeded the maximum flow that could make it through the above-water holes.

Hopefully the new sealing and (not for CO2 - see update above) check valve will do the trick.

@“Martin”#p96 A correct O2 sparging will depend on the size of bubbles (the littler the better) and the purging time (5-10min should be OK). Considering the size of the pioreactor, the tube you are using for media recirculation should be enough.