Table 3 Equipment maintaining continuous culture to sustain continuous evolution of microbial cell factories

Flask

ml-l

Single

No

N/A

N/A

Bulk

manual

48 h

Shaken in shaker incubator to ensure well gas exchange. Dilution is performed when required

N/A

Chemostat

l

Single

No

Flow rate

N/A

Bulk

active pump

Several days

Continuous dilution is achieved by continuous flow of liquid medium in and out of the vessel

[99,100,101]

In-vial continuous cultivation system

< 15 ml

Several vials

Yes

Inhibitor concentration, cell concentration, stirring speed, temperature^a

Optical density, fluorescence

Bulk

Passive pressure, millifluidic^a, valve

24 h

(Continuous cultured for 144 h)

Dilution triggered by defined parameter, while the liquid flow out by passive pressure. Parallelisation is achieved through miniaturisation and parallel control of multiple vials. However, biofilm formed in the vials and human intervention is required every 24 h

[103,104,105, 109]

Microfluidic-based continuous cultivation system

nl-ml

6 channels

No

Inhibitor concentration, cell concentration

Optical density, microscopy

Single-cell

microfluidic, valve

> 500 h

Continuous cultivation by circulating the culture around microfluidic channel. Biofilm is removed and the culture is diluted by frequently flushing a segment of the channel with lysis buffer and culture medium

[113, 114]

Droplet-based continuous cultivation system

nl-μl

~ Thousands of droplets

Yes

Inhibitor concentration, cell concentration, temperature

Optical density, fluorescence

Single-cell

Millifluidic, valve

Months

Encapsulation of microbial single-cell into droplet, and exchange medium through droplet breaking and pico-injection

[123, 124]