Abx pentra glucose hk cp reagent

A single colony was used to inoculate 100 ml of C/N 75 medium. After 24 h growth at 30 °C and 250 rpm agitation the inoculum was transferred into a 1,25 L fermenter.
Fed-batch fermentation was performed in 1 l BioFlo 115 fermenter using the C/N 75 medium. After depletion of the initial glucose concentration a glucose feed of 1,1 g/h was established. The pH was controlled with 2 M NaOH or 1 M H₃PO₄. During the fermentation process 4 ml samples were taken using an automatic sampler (Gilson art. no. F203B). Collected samples were cooled to 1 °C to quench the metabolism. Samples were used for dry weight determination and the presence of residual glucose was analysed on a Cobas Mira Plus using the Horiba ABX Pentra Glucose HK CP reagent (art. no. A11A01667).

Glycogen was analysed according to a modified method as described by Aklujkar et al. [44 (link)]. To 30 mg of dry weight cells 500 μl of 2 M NaOH was added followed by boiling for 1 h. To neutralize this mixture, 60 μl of 9 M H₂SO₄ and 600 μl 1 M NaAc/HAc buffer (pH 4,5) were added. To 400 μl of sample, 50 units of glucoamylase (Novozymes art. no. NS22035) were added and incubated at 50 °C for 1 h. The final glucose concentration of the mixture was measured with and without amyloglucosidase treatment on a Cobas Mira Plus using the Horiba ABX Pentra Glucose HK CP reagent (art. no. A11A01667). Maltose was used as a positive control to check the activity of the amyloglucosidase.

Batch fermentations were performed in 7 l BioFlo115 fermenters using the C/N 5 medium. The pH was controlled with 2 M NaOH or 1 M H₃PO₄. During the fermentation process 4 ml samples were taken using an automatic sampler (Gilson art. no. F203B). Collected samples were cooled to 1 °C to quench the metabolism. Samples were used for dry weight determination and the presence of residual glucose was analysed on a Cobas Mira Plus using the Horiba ABX Pentra Glucose HK CP reagent (art. no. A11A01667).