Preparation of inoculum
Cove-N-Gly plates were inoculated with spore suspension from the −80 °C stock of each strain.
The plates were incubated at 30 °C for 7 days. Subsequently, the spores were harvested by addition of 10 mL of 0.01 % Tween 80 solution.
The spore suspension was used to inoculate G2-GLY medium with a final concentration of 6×109 spores L−1. The preculture was incubated after shaking at 250 rpm in a 500-mL shake flask without baffles for 24 h at 30 °C.
Thereafter, the main cultures for malic acid production in fermentors or shake flasks were inoculated with 10 % of the final volume of the preculture broth.