Rapid sampling, cell inactivation and evaluation of low extracellular glucose concentrations during fed-batch...

A method for rapid extracellular sampling, cell inactivation and handling of a large number samples has been developed and evaluated. This method might be used during experiments, where concentrations of extracellular components in the range of some milligrams per litre has to be evaluated. The analysis should be performed by enzymatic or colorimetric analysis and is shown to be suitable for measuring low nutrient concentrations 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 than 0.15 s, a time during which negligible glucose is shown to be consumed from the sample. The glucose consumption is stopped by a rapid pH decrease using perchloric acid in a defined concentration that depended on the organism studied. This concentration was chosen in order to avoid cell lysis that could affect the glucose concentration by the intracellular release of glucose containing compounds and thereby expose them to the acid. It was seen that the sample mixed rapidly with the acid and that no hydrolysis of glucose related compounds interfered with the analysis. The samples were neutralised and the precipitation centrifuged 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 handling of a large number of samples was also devised which is based on measurements on microtiter plates and allows samples to be evaluated with a high statistical accuracy with minimum sample waste in a short time. The ability of the method is shown by a study of the metabolic response of a shift in glucose feed, as followed by glucose analysis.