2.4 Automatic adaptive laboratory evolution
The automatic ALE experiment for high crude glycerol tolerance was carried out continuously in a novel system developed by our group as shown in Fig. 1 . After pre-cultivation in pure glycerol medium, an active culture was inoculated into an anaerobic bottle with a starting crude glycerol concentration of 30 g/L. The initial OD was set at 0.5 by adjusting the inoculum size. Cultivation was maintained at 35°C with a constant stirring at 50 rpm using a magnetic stirring bar. To realize a real-time measurement of OD600, 2 mL of the culture in the anaerobic bottle were automatically transferred to a photometer (Ultrospec 10, Biochrom) for OD detection every 2 min. After the OD value was recorded by the computer, the culture in the detection cuvette was transferred back to the anaerobic bottle to maintain a constant working volume of 50 mL. Once the OD600 reached 1.5, the inlet flow of fresh medium (with the same crude glycerol concentration) and the outlet flow (waste) of culture were initiated simultaneously to dilute the culture until the OD value was reduced back to 0.5, and then the next cycle of adaptation continued. The time interval (△T) for each adaptation cycle was recorded, and the concentration of crude glycerol in the fresh medium was increased by 10 g/L each time when the △T maintained constant for more than 10 cycles. After more than 100 adaptation cycles from 30 to120 g/L crude glycerol, an end-point adapted strain (termed asC. pasteurianum G8) which was fast-growing without any lag phase in 120 g/L crude glycerol, was selected for further fermentation experiments.