Optimisation of the preservation of microbial cell banks for enhanced fermentation process performance

Abstract

This work discusses optimisation of the cryopreservation of Bacillus licheniformis cell banks, used as inoculum for α-amylase producing 5 L batch fermentations. The effect of the presence of various cryopreservants including glycerol, Tween 80 and dimethyl sulphoxide on final fermentation performance measured by biomass and α-amylase concentration was investigated using optical density, dry cell weight, colony forming units, and multi-parameter flow cytometry. The application of multi-parameter flow cytometry using the fluorophores DiBac$_4$(3) and PI allowed real time viability measurements of individual microbial cells to be monitored before and after cryopreservation and during the fermentation process; viability here being defined as a cell having an intact and fully polarised cytoplasmic membrane. It was found that the concentration and type of cryopreservant used had a significant effect on microbial cell physiology and population heterogeneity during resuscitation recovery immediately after thawing. Cell banks prepared with Tween 80 were fastest to recover after freezing in comparison to cell banks prepared with dimethyl sulphoxide which showed the slowest growth rates. Interestingly cells preserved in glycerol recovered at a similar rate to cells frozen without cryopreservant. Despite different responses to the freezing process when each cell bank was used as inoculum for 5 L batch fermentations very little difference was noticed in overall process performance with respect to α-amylase production, growth rate and final biomass concentration