Laboratory scale fed-batch cultivations
The chemostat cultivations indicated an optimal F:G ratio in the feed of 5 mol/mol. This formed the basis of the experimental design of a fed-batch protocol in which the positive effect of formic acid co-feeding on the yield was to be confirmed in this industrially more relevant fermentation mode. Four variations of the fed-batch protocol were tested on laboratory scale, with a fixed feeding rate and F:G ratios that ranged from 0 (experiment LF1), 3 (LF2), 5 (LF3) to 7 (LF4) by replacing part of the water in the carbon feed solution by formic acid. A dynamic fermentation process model (Supplementary Materials 1) was developed and applied to quantitatively predict broth weight development, OUR, as well as other fermentation variables, which allowed to design the experiments such that they would fit the experimental set-up.
The laboratory scale fed batch process proved successful. After the initial batch phase on glucose only, Y. lipolytica readily consumed the mixed glucose/ formic acid feed when it was dosed at a carbon-limiting rate. Throughout the fermentations, supernatant samples were taken and analysed for residual formic acid, and the concentrations were always low (<0.06 g/l) or below the detection limit.
Figure 4 shows the O2 uptake rate profiles of the 4 laboratory scale fermentations with the batch phases ending between 10-15h, followed by the carbon-limited fed batch phases. The decrease of the OUR after ~55h for LF1 and after ~60h for LF2 is caused by O2 transfer limitation of the fermenter. This limitation resulted from the increasing biomass concentration which in turn led to broth viscosity, a factor that was not accounted for in the dynamic process model. Until the onset of the late O2 transfer limitation, the experimental data quantitatively correspond with the simulated fermentations using the dynamic process model (see Supplementary Materials 1).