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).