Propionic acid is widely used in food and dairy industries. The demand of propionic acid for use as a natural preservative in foods and grains is high. Fermentation by propionibacteria produces propionic acid from sugars; however, the fermentation suffers from low propionic acid production due to by-product formation and strong propionate inhibition on cell growth and the fermentation.
In this work, fed-batch fermentation of glucose by Propionibacterium acidipropionici immobilized in a fibrous-bed bioreactor (FBB) with a high cell density (>45 g/L) produced a high propionate concentration of 72 g/L and a high propionate yield of up to 0.65 g/g. A mutant with improved propionate tolerance was obtained by adaptation in the FBB, resulting in significant physiological and morphological changes. The mutant was less sensitive to propionate inhibition and had a higher saturated fatty acid content in the cell membrane and a slimmer shape with an increased specific surface area.
Metabolic stoichiometric analysis was applied to quantitatively describe the global cellular mechanism in propionic acid fermentation. By feeding carbon sources with different oxidation states, different fermentation end-product compositions were obtained, indicating different controlling mechanisms involving various acid-forming enzymes with significant changes in their activities and overall protein expression pattern. The metabolic pathway generally shifted toward more propionate formation with a more-reduced substrate.
Gene inactivation via gene disruption and integrational mutagenesis was used to knock out the acetate kinase (ack) gene with the goal of eliminating acetate formation and further enhancing propionate production. Mutants were obtained by transforming the cells with a partial ack gene introduced either as a linear fragment with a tetracycline resistance cassette within the partial ack gene or in a non-replicative integrational plasmid containing the tetracycline resistance cassette. The ack inactivation in the mutants showed a profound impact on cell growth rate. Compared to the wild type, the ack-deleted mutants achieved ~10% increase in propionate yield and ~10% decrease in acetate yield.
The FBB, the knowledge of the underlying mechanism in controlling propionic acid fermentation, and the mutants obtained in this research should allow us to develop an economical bioprocess for the production of propionic acid from sugars.