RAPID SAMPLING, CELL INACTIVATION AND EVALUATION OF LOW EXTRACELLULARGLUCOSE-CONCENTRATIONS DURING FED-BATCH CULTIVATION

A method for rapid extracellular sampling, cell inactivation and handl ing of a large number samples has been developed and evaluated. This m ethod might be used during experiments, where concentrations of extrac ellular components in the range of some milligrams per litre has to be evaluated. The analysis should be performed by enzymatic or colorimet ric analysis and is shown to be suitable for measuring low nutrient co ncentrations present in fed-batch cultivation. The test organisms were Escherichia coli and Saccharomyces cerevisiae and the model substance was glucose. Using this technique, the sample can be taken in less th an 0.15 s, a time during which negligible glucose is shown to be consu med from the sample. The glucose consumption is stopped by a rapid pH decrease using perchloric acid in a defined concentration that depende d on the organism studied. This concentration was chosen in order to a void cell lysis that could affect the glucose concentration by the int racellular release of glucose containing compounds and thereby expose them to the acid. It was seen that the sample mixed rapidly with the a cid and that no hydrolysis of glucose related compounds interfered wit h the analysis. The samples were neutralised and the precipitation cen trifuged to minimise the effect on the chosen enzymatic analysis. This analysis was modified and the accuracy determined in order to analyse concentrations in the milligram range. A method to increase the handl ing of a large number of samples was also devised which is based on me asurements on microtiter plates and allows samples to be evaluated wit h a high statistical accuracy with minimum sample waste in a short tim e. The ability of the method is shown by a study of the metabolic resp onse of a shift in glucose feed, as followed by glucose analysis.