Evaluation of a pH-stat feeding strategy on the production and recovery of Fab? fragments from

A comparison between two feeding strategies in a gas blending fermentation process to produce Fab’ by Escherichia coli was carried out at 20L scale. A pH-stat feeding strategy was proposed to address the issue of glycerol oscillations that occur in repeated batch fermentations. This feeding strategy was based on direct coupling of growth and concomitant production of H+ and this led to a constant minimum concentration of glycerol (main C-source) in the induction period. This change of environment resulted in a two-fold increase in the production of Fab’ compared to those obtained using a repeated batch strategy. A glycerol yield coefficient (YX/glycerol in gcell/gglycerol) and product yield on biomass (YP/X in mgproduct/gcell) of 0.39 and 5.91, respectively, were estimated for the pH-stat feeding fermentation. By contrast, YX/glycerol and YP/X were found to be 13 and 45% lower, respectively, for the repeated batch process. Therefore, considerable improvements in glycerol utilisation and product formation have been obtained using the pH-stat feeding strategy. Product localisation in the cell periplasm of >90% was obtained in both processes and a difference of product loss in the centrifugation step of less than 1% was observed. Overall, a method to improve production of Fab’ fragments without compromising primary recovery of cells by maintaining constant low levels of glycerol has been developed.