Continuous culture using pioreactor

[Subhashree]

Hello everyone,

I’m looking to connect with anyone running continuous cultures using a Pioreactor. I’ve been encountering an issue over the past few runs and would appreciate any insights.

I am cultivating Acinetobacter, and in shake flask experiments the doubling time is typically around 30 minutes. However, when using the Pioreactor, the doubling time increases to approximately 2 hours.

I am using LB medium, so I don’t suspect a media-related problem. I initiate the continuous culture once the OD reaches ~1, with a flow rate of 1 mL/hour (Pioreactor volume: 10 mL). I consistently observe that the OD begins to decrease once the feed starts.

Has anyone experienced a similar issue? I would greatly appreciate any insights or shared experiences. I’m interested in knowing if continuous culture has worked for anyone. If yes, could you share details about your setup?

I dont use air bubbler, so no air flow to the pioreactor. I am suspecting oxygen limitation.

[CamDavidsonPilon]

@Subhashree

I consistently observe that the OD begins to decrease once the feed starts.

It sounds like your running a chemostat at dilution rate 0.1/h, and you are washing out the culture => your cultures growth is less than 0.1/h. That’s pretty low.

What is your RPM during the experiment? The Pioreactor is limited on oxygen without additional hardware - adding oxygen might help yes.

---

Are you starting the chemostat early, mid, or late in the experiment?

[Subhashree]

Hey Cam,

Adding oxygen definitely helped, but I’m facing a couple of other issues.

I’m operating the Pioreactor in chemostat mode with a media flow rate of 1.2 mL per hour, and the waste multiplier is on. The OD is stable now.

However, after a few hours, the calculated vial volume exceeded 18 mL. The logs show “vial maximum volume reached,” so feeding stopped even though the waste pump was running. I’ve attached the logs; could you please take a look?

This hasn’t happened before.
export_20260304192719.zip (216.5 KB)

[CamDavidsonPilon]

Hm, looking now. Can you tell me what the value you entered for this field is?

[Subhashree]

Initial Volume 12ml , Max volume 18ml

[CamDavidsonPilon]

So the issue is 18ml. That’s at the safety margin, and will halt pumping (this is a hardcoded constant in the system).

That field is perhaps confusing, and I’ve tried many iterations to make it more clear. That field should be the volume s.t. your waste tube is placed at the liquid-air interface in the vial with that volume.

Is your waste tube indeed that high in the vial? Do you expect to be have 18ml of culture at the end?

[Subhashree]

@CamDavidsonPilon

hat’s at the safety margin, and will halt pumping (this is a hardcoded constant in the system).

I fed the media at a rate of 0.2 mL every 10 minutes, which equals 1.2 mL per hour in total. The waste tube was not positioned very high; it was just touching the liquid level when the pioreactor volume was 12 mL. Therefore, as long as the waste pump is running, the volume inside the pioreactor vial should remain at 12 mL.

However, the pioreactor does not actually sense the liquid level, right? It only estimates the vial volume based on pump speeds. So if the waste pump is running and its flow rate is equal to or higher than the media inflow (for example, when the waste multiplier is active), then the measured vial volume should not increase, correct?

[CamDavidsonPilon]

@Subhashree

the volume inside the pioreactor vial should remain at 12 mL.

Yes, so the correct value of the field in the form should have been 12ml, and not 18ml (you can see why I am having trouble explaining this!)

However, the pioreactor does not actually sense the liquid level, right?

Correct, we just infer it from software’s events.

So if the waste pump is running and its flow rate is equal to or higher than the media inflow (for example, when the waste multiplier is active), then the measured vial volume should not increase, correct?

This is correct, but not what is used in practice.

In practice, assuming the correct values “Initial Volume 12ml , Max volume 12ml”, we start with:

12ml

then when the chemostat runs the first time (or any dosing automation), we add 0.2ml:

12.2ml

and then remove 0.2ml

12.0ml

and then remove 0.4ml more (this is effectively over-removing liquid, in case waste pump falls behind or whatever). But since the waste tube is at 12ml, this does nothing (in software):

12.0ml

---

However, if your settings are “Initial Volume 12ml , Max volume 18ml”, then it looks like

12.0ml

Add the first dose of 0.2ml

12.2ml

Remove 0.2ml, but the software thinks your waste pump is higher up at 18ml in the vial, so the software doesn’t decrement:

12.2ml

Remove 0.4ml, but same logic:

12.2ml

…

And you can see why your computed vial volume keeps increasing.

[CamDavidsonPilon]

I’ve added a diagram to make these fields more clear