Interaction of cultural conditions and end-product distribution in Bacillus subtilis grown in shake flasks

Summary A culture of Bacillus subtilis, in which the relative production of acetoin (Ac) and butanediol (Bu) is highly sensitive to oxygen tension as well as to mixing conditions, was used to evaluate several culture conditions in 500-ml shake flasks. The concentration ratio of these metabolites (Ac/Bu) produced in a defined period of culture time was used as a parameter for comparative purposes. The influence of working volume, shaking speed, broth viscosity and the presence of baffles were evaluated. Using unbaffled flasks it was found that working volume had the most influence on oxygenation in shake flasks, especially below 10%, where differences in Ac/Bu ratios up to ten times could be measured. Shaking speed played an important role only at values higher than 400 rpm or when small working volumes were used. The addition of xanthan gum decreased the Ac/Bu ratio nearly four times under equivalent working conditions and also diminished the influence of shaking speed. In general, Ac/Bu was higher when sulphite oxygen transfer rate (OTR) values were higher. However, the test culture was able to detect differences which were not evident using the OTR method. Comparing Ac/Bu ratios in stirred fermentors from the literature, it seems that similar oxygenation conditions can be reached in non-baffled shake flasks only at very high shaking speeds using small working volumes. With baffled flasks, our data suggest that better oxygenation and mixing can be achieved in shake flasks if compared with those obtained in stirred fermentors at conventional power inputs.