Each strain was grown on D-glucose during the batch phase for around 24 h, until the glucose was depleted.
Figure 6b shows that by operating at D = 0.01 h−1 after the initial batch phase for approximately 90 h, then increasing D incrementally after holding times of approximately 100 h, results in a steady increase in CSA up to 36.6 g L−1, followed by a stable value of 35.3 g L−1 on average.
Fed-batch cultures are usually split into two stages: batch phase for cell growth and fed-batch phase for PHB accumulation.
The observation that the apparent μ of the cells was different to the set μ during the non-induced fed-batch phase for both cultures may also be attributed to cell lysis since the Kalman filter, regulating the feed according to intact cells, did not respond to cell lysis.
At this stage of the culture, the biomass dry weight concentrations were between 15 to 18 g L-1 and remained steady during the whole fed-batch phase for all cultures whatever conditions were tested.
During the fed-batch phase, conditions were the same as for the batch phase except that the airflow was adjusted to remain at 1 vvm, i.e., increased with culture volume.
Based on the data from the batch phase, the maximum specific growth rates (μmax) for both strains (one expressing 9704440 and a negative control) were calculated.
After the batch and glycerol fed-batch phases for biomass build-up, the wet biomass concentration in the fermentation of PMO-01867, PMO-02916, PMO-03328 and PMO-08760 reached 349, 290, 371 and 342 g L-1, respectively.
For bioprocess control, a batch phase involving the fermentation basal salt medium containing 40 g/L glycerol (PFPG, Invitrogen; Liu et al. 2018) was first conducted and maintained for approximately 18 h.
