Difficutly with Dilution (rate)

Using the Pioreactor
1

I have been trying to figure out the max specific growth rate of a microbe by stepping the dilution rate, or more recently the target OD in turbidostat mode.

I have dilution rates well above what a max specific growth rate should be and at time i haven’t been able to get the OD down with increased dilution rates.

  1. It’s possible my OD is off, as my blanking was wrong and that .3 is actually blank and washout. regardless…
  2. The dilution rates are WAY too high at nearly all points. My only guess is that the reactors can’t properly mix and dilute, so the fresh media that is added on top is mostly what is getting removed, keeping the bottom of the reactor from getting diluted and causing way more media cycling than should be necessary. In that case the dilution rate will not equal the growth rate.

April 18 pioreactor 3
April 18 pioreactor 31880×1351 254 KB

2

To address point 2., there is a configuration parameter pause_between_subdoses_seconds that you can increase to provide more time for mixing between media and waste pump. I actually haven’t tested this parameter much, and arbitrarily picked a default of 5 sec, which I figured would be more than enough.

3

There is also a limit based on your dose volume and pump speed too. I had run into the maxium effective dilution rate because the dose was only .5ml and at top speed it was cycling every 45 seconds. I set my profile script then step up the dose to 1ml. Still I’m not sure how long it will take to mix well. Also not sure what the most effective dose size would be.

I could consider running actual volume above the waste tube and removing media first.

4

If you want more dosing per minute, another parameter to modify is waste_removal_multiplier. This parameter controls the second waste dose. For example, running a chemostat with 0.5 ml volumes:

  1. 0.5 ml is added via media_pump
  2. 0.5 ml is removed via waste_pump
  3. waste_removal_multiplier * 0.5 ml is removed again via waste_pump

This is to prevent overflow if the waste-pump is under-pumping (else the delta builds up, and overflows). By default, the waste_removal_multiplier is 2.0. You can probably change this to 1.0, or even 0.5. This will allow you to make more doses per unit time. 0.0 is a valid value, too, but is risky.

(Note, this system of running the waste pump again usually prevents ideas like “running actual volume above the waste tube”, since eventually the we converge to the waste tube from this double removal.)

1.0ml dose size seems like another obvious optimization too.