Metabolic studies of γ-polyglutamic acid production in Bacillus licheniformis by small-scale continuous cultivations

For the cultivation of microorganisms shake flasks are frequently used. The development of the broth viscosity during the cultivation is usually not paid special attention to. However, changes in viscosity may have a severe impact on shake flask cultivations, ranging from effects on mass and heat transfer to the occurrence of out-of-phase conditions. Significant changes in viscosity were observed during batch cultivations with Bacillus licheniformis on defined mineral medium. The formed biopolymer was identified to be γ-polyglutamic acid (γ-PGA), a polymer, Bacillus species are known to excrete as a side-product. γ-PGA leads to increasing viscosity and, thus, has critical influences on shake flask cultivations. Consequently, the oxygen transfer is strongly influenced by viscosity and, therefore, γ-PGA formation. Furthermore, γ-PGA has chelating characteristics, which might affect nutrient and trace element availability, possibly resulting in substrate limitations. In total, these influences lead to undefined conditions in the fermentation. Therefore, the trigger for γ-PGA production was investigated. By performing continuous cultivation experiments as well as pulse experiments, catabolite controlled overflow was identified as one of the key triggers for γ-PGA production. The results suggest, that catabolite controlled overflow leads to inhibition of the 2-oxoglutarate complex, resulting in accumulation of 2-oxoglutarate and a redirection of metabolic flux toward glutamic acid and finally to γ-PGA. Additionally, oxygen limitations were determined to further potentiate γ-PGA production. As a result, fermentation conditions favoring catabolite controlled overflow as well as oxygen limitation need to be strictly avoided in order to assure defined conditions at low viscosity throughout the fermentation.

► Viscosity increase seen in protease production with γ-PGA forming B. licheniformis.
► High viscosity severely affects fermentation, results in undefined conditions.
► Continuous cultivation and pulse experiments were performed.
► Excess carbon and oxygen limitation have similar effect on viscosity.
► Key trigger for γ-PGA production is catabolite controlled overflow.