Simple and rapid gas-chromatographic determination of volatile metabolites in fermentation broths

Bacillus subtilis K (AJ 1992) responds very rapidly to variations in oxygen supply, excreting volatile acetoin and meso-2,3-butanediol; their relative amounts are determined by the supply of dissolved oxygen. A complex medium was used to grow B. subtilis. Initially, packed-column gas chromatography was used. The samples were centrifuged and then directly injected. The precision was about 5%. A complete analysis lasted about 22 min. To get better results a wide-bore capillary column was used. Problems with reproducibility were encountered when split injection was used, whereas a packed-column inlet and autosampler/injection gave precise results for clean samples (s.d. 0.7% for acetoin, 3.3% for 2,3-butanediol). Slight nonlinearities were observed for butanediol below 0.5 gl −1. A complete analysis was then possible within 10 min. Both types of column allowed meso- and d,l-2,3-butanediol to be distinguished. High concentration of molasses and larger amounts of silicone antifoam agent caused peak area drift, especially for butanediol. Data evaluation based on 2 to 3 reference injections, and linear interpolation allowed an accuracy better than ± 5% for both volatile components. Total culture broth including cells could be injected directly. Samples containing xanthan gum could be injected without any pretreatment. The method developed may be applicable in many bioprocesses where volatile components have to be determined.